TGR5 modulators and methods of use thereof

ABSTRACT

The application relates to compounds of formula A: 
                         
or a salt, solvate, ester, tautomer, amino acid conjugate, or metabolite thereof. The compounds of formula A are TGR5 modulators useful for the treatment of various diseases, including metabolic disease, inflammatory disease, autoimmune disease, cardiac disease, kidney disease, cancer, and gastrointestinal disease.

FIELD OF THE APPLICATION

The application relates to compounds that modulate TGR5 and compositionsuseful in methods for the treatment and/or prevention of variousdiseases.

BACKGROUND

TGR5 is a G-protein-coupled cell-surface receptor that is responsive tobile acids (BAs). The primary structure of TGR5 and its responsivenessto bile acids has been found to be highly conserved in human, cow,rabbit, rat, and mouse, and thus suggests that TGR5 has importantphysiological functions. TGR5 is widely distributed in not only lymphoidtissues but also other tissues. High levels of TGR5 mRNA have beendetected in placenta, spleen, and monocytes/macrophages. Bile acids havebeen shown to induce internalization of the TGR5 fusion protein from thecell membrane to the cytoplasm (Kawamata et al., 2003, J. Bio. Chem.278, 9435). TGR5 has been found to be identical to hGPCR19 (Takeda etal. 2002, FEBS Lett. 520, 97).

TGR5 is associated with the intracellular accumulation of cAMP, which iswidely expressed in diverse cell types. While its activation inmacrophages decreases pro-inflammatory cytokine production (Kawamata etal., 2003, J. Bio. Chem. 278, 9435), the stimulation of TGR5 by BAs inadipocytes and myocytes enhances energy expenditure (Watanabe et al.,2006, Nature 439, 484). This latter effect involves the cAMP-dependentinduction of type 2 iodothyronine deiodinase (D2), which, by locallyconverting T4 into T3, gives rise to increased thyroid hormone activity.Consistent with the role of TGR5 in energy metabolism, female TGR5knock-out mice show a significant fat accumulation with body weight gainwhen challenged with a high fat diet, indicating that the lack of TGR5decreases energy expenditure and elicits obesity (Maruyama et al., 2006,J. Endocrinol. 191, 197). In addition and in line with the involvementof TGR5 in energy homeostasis, bile acid activation of the membranereceptor has been reported to promote the production of glucagon-likepeptide 1 (GLP-1) in murine enteroendocrine cell lines (Katsuma, 2005,Biochem. Biophys. Res. Comm. 329, 386). Thus, TGR5 is an attractivetarget for the treatment of diseases (e.g., obesity, diabetes andmetabolic syndrome).

In addition to the use of TGR5 agonists for the treatment and preventionof metabolic diseases, compounds that modulate TGR5 are also useful forthe treatment of other diseases, e.g., central nervous diseases as wellas inflammatory diseases (WO 01/77325 and WO 02/84286). Moreover,modulators of TGR5 can be used in methods of regulating bile acid andcholesterol homeostasis, fatty acid absorption, and protein andcarbohydrate digestion.

Recently, 23-alkyl-substituted and 6,23-dialkyl-substituted derivativesof chenodeoxycholic acid (CDCA), such as6α-ethyl-23(S)-methyl-chenodeoxycholic acid, have been reported aspotent and selective agonists of TGR5 (Pellicciari et al., 2007, J. Med.Chem. 50, 4265). TGR5 agonists have also provided for the first time apharmacological differentiation of genomic versus nongenomic effects ofBAs and allowed for informative structure-activity relationship studies.In this context, the availability of more potent and selective TGR5modulators is necessary to further identify additional featuresaffecting receptor activation and to characterize the physiological andpharmacological actions of this receptor in order to better understandits relationship to the prevention and treatment of diseases.

Thus, there is a need for the development of TGR5 modulators for thetreatment and/or prevention of various diseases. The present applicationhas identified compounds that modulate TGR5 as well as methods of usingthese compounds to treat or prevent diseases in which TGR5 is involved.

SUMMARY

The present application relates to TGR5 modulators and their use totreat and/or prevent various diseases. In one aspect, the applicationrelates to a compound having formula A:

or a pharmaceutically acceptable salt, solvate, ester, tautomer, aminoacid conjugate, or metabolite thereof, wherein n, R₁, R₂, and R₃ can beselected from the respective groups of chemical moieties later definedin the detailed description.

In another aspect, the application relates to a pharmaceuticalcomposition comprising a compound of the application or apharmaceutically acceptable salt, solvate, ester, tautomer, amino acidconjugate, or metabolite thereof, and at least one pharmaceuticallyacceptable excipient.

In yet another aspect, the application relates to a method of treatingor preventing a disease or disorder in a subject, comprisingadministering to the subject an effective amount of a compound of theapplication, or a pharmaceutically acceptable salt, solvate, ester,tautomer, amino acid conjugate, or metabolite thereof.

In yet another aspect, the application relates to a compound of theapplication, or a pharmaceutically acceptable salt, solvate, ester,tautomer, amino acid conjugate, or metabolite thereof, for use in amethod of treating or preventing a disease or disorder in a subject.

In yet another aspect, the application relates to use of a compound ofthe application, or a pharmaceutically acceptable salt, solvate, ester,tautomer, amino acid conjugate, or metabolite thereof, in themanufacture of a medicament for treating or preventing a disease ordisorder in a subject.

In one aspect, TGR5 is involved in the disease or disorder. In oneaspect, TGR5 plays a role in the activation/upregulation of the cellularpathway which results in the disease or disorder. In another aspect,TGR5 plays a role in the de-activation/downregulation of the cellularpathway which results in the disease or disorder. In a furtherembodiment, the disease or disorder is selected from a metabolicdisease, an inflammatory disease, an autoimmune disease, a cardiacdisease, a kidney disease, a gastrointestinal disease, a pulmonarydisease, and a cancer.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this application belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent application, suitable methods and materials are described below.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the present application. Inthe case of conflict, the present specification, including definitions,will control. In addition, the materials, methods, and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the application will be apparent fromthe following detailed description and claims.

DETAILED DESCRIPTION Definitions

For convenience, certain terms used in the specification, examples andclaims are collected here.

As used herein, “BA” means bile acid and bile acid derivatives. Bileacids are steroid carboxylic acids derived from cholesterol. The primarybile acids are cholic and chenodeoxycholic acids. In the body, theseacids are conjugated with glycine or taurine before they are secretedinto the bile.

“Alkyl” refers to saturated aliphatic groups, including straight chainalkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl), branched chain alkyl groups (e.g., isopropyl,tert-butyl, isobutyl). In certain embodiments, a straight chain orbranched chain alkyl has six or fewer carbon atoms in its backbone,referred to as “lower alkyl” (e.g., C₁-C₆ for straight chain meaning 1,2, 3, 4, 5, or 6 carbon atoms, C₃-C₆ for branched chain meaning 3, 4, 5,or 6 carbon atoms). In some examples, a straight chain or branched chainalkyl has four or fewer carbon atoms in its backbone. In furtherexamples, a straight chain or branched chain alkyl has three or fewercarbon atoms in its backbone.

The term “substituted alkyl” refers to an alkyl moiety having asubstituent replace one or more hydrogen atoms on at least one carbon ofthe hydrocarbon backbone. Such substituents can include, for example,halogen, hydroxyl, alkoxyl, alkylcarbonyl, alkoxycarbonyl, carboxylate,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, cyano, amino,nitro, and cyano.

The term “alkoxy” or “alkoxyl” includes alkyl, alkenyl, and alkynylgroups covalently linked to an oxygen atom. Examples of alkoxy groups(or alkoxyl radicals) include methoxy, ethoxy, isopropyloxy, propoxy,butoxy, and pentoxy groups.

The term “ester” refers to moieties which contain a carbon or aheteroatom bound to an oxygen atom which is bonded to the carbon of acarbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

The term “halogen” includes fluorine, bromine, chlorine, iodine, etc.The term “perhalogenated” generally refers to a moiety wherein allhydrogens are replaced by halogen atoms.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R₁ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “unstable functionality” refers to a substitution pattern thatcontains a labile linkage, e.g., a functionality or bond that issusceptible to hydrolysis or cleavage under physiological conditions(e.g., aqueous solutions in the neutral pH range). Examples of unstablefunctionalities include acetals and ketals.

Additionally, the compounds of the present application or salts thereof,can exist in either hydrated or unhydrated (the anhydrous) form, or assolvates with other solvent molecules. Nonlimiting examples of hydratesinclude monohydrates, dihydrates, etc. Nonlimiting examples of solvatesinclude ethanol solvates, acetone solvates, etc.

“Solvates” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate, when the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one of the substances in whichthe water retains its molecular state as H₂O, such combination beingable to form one or more hydrate.

It will be noted that the structure of some of the compounds of theapplication include asymmetric carbon atoms. It is to be understoodaccordingly that the isomers arising from such asymmetry (e.g., allenantiomers and diastereomers) are included within the scope of theapplication, unless indicated otherwise. Such isomers can be obtained insubstantially pure form by classical separation techniques and bystereochemically controlled synthesis. Enantiomers (R- andS-configurations) are named according to the system developed by R. S.Cahn, C. Ingold, and V. Prelog.

Further, the structures and other compounds discussed in thisapplication include all atropic isomers thereof. Atropic isomers are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture.However, as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solid form,usually one tautomer predominates. In solutions where tautomerization ispossible, a chemical equilibrium of the tautomers will be reached. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent and pH. The concept of tautomers that areinterconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—-CHO) in a sugar chain molecule reacting with one ofthe hydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose. Common tautomeric pairs are:ketone-enol, amide-nitrile, lactam-lactim, amide-imidic acid tautomerismin heterocyclic rings (e.g., in nucleobases such as guanine, thymine andcytosine), amine-enamine and enamine-enamine.

It is to be understood that the compounds of the present application maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present application, and the namingof the compounds does not exclude any tautomer form.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar to or comparable in function and appearance tothe reference compound.

As defined herein, the term “derivative”, e.g., in the term “bile acidderivatives”, refers to compounds that have a common core 4-memberedring structure, and are substituted with various groups as describedherein.

As defined herein, the term “metabolite”, e.g., in the term “bile acidmetabolites”, refers to glucuronidated and sulphated derivatives of thecompounds described herein, wherein one or more glucuronic acid orsulphate moieties are linked to the bile acid compounds describedherein. Glucuronic acid moieties may be linked to the bile acidcompounds through glycosidic bonds with the hydroxyl groups of the bileacid compounds (e.g., 3-hydroxyl, 7-hydroxyl, 12-hydroxyl, and/or15-hydroxyl). Sulphated derivatives of the bile acid compounds may beformed through sulfation of the hydroxyl groups (e.g., 3-hydroxyl,7-hydroxyl, 12-hydroxyl, and/or 15-hydroxyl). Examples of bile acidmetabolites include, but are not limited to, 3-O-glucuronide,7-O-glucuronide, 12-O-glucuronide, 15-O-glucuronide,3-O-7-O-glucuronide, 3-O-12-O-glucuronide, 3-O-15-O-glucuronide,7-O-12-O-glucuronide, 7-O-15-O-glucuronide, 12-O-15-O-glucuronide,3-O-7-O-12-O-glucuronide, 3-O-7-O-15-O-glucuronide, and7-O-12-O-15-O-glucuronide, of the bile acid compounds described herein,and 3-sulphate, 7-sulphate, 12-sulphate, 15-sulphate, 3,7-bisulphate,3,12-bisulphate, 3,15-bisulphate, 7,12-bisulphate, 7,15-bisulphate,3,7,12-trisulphate, 3,7,15-trisulphate, 7,12,15-trisulphate, of the bileacid compounds described herein.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres includeacyl sulfonimides, tetrazoles, sulfonates, and phosphonates. See, e.g.,Patani and LaVoie, Chem. Rev. 96, 3147-3176 (1996).

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the application wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic,glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodide, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic,phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic,succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic.

The pharmaceutically acceptable salts of the present application can besynthesized from the parent compound that contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, non-aqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 18th ed., Mack Publishing Company, Easton, Pa.,USA, page 1445 (1990).

As used herein, the term “amino acid conjugates” refers to conjugates ofthe compounds of the application with any suitable amino acid. Taurine(NH(CH₂)₂SO₃H), glycine (NHCH₂CO₂H), and sarcosine (N(CH₃)CH₂CO₂H) areexamples of amino acid conjugates. Suitable amino acid conjugates of thecompounds have the added advantage of enhanced integrity in bile orintestinal fluids. Suitable amino acids are not limited to taurine,glycine, and sarcosine. The application encompasses amino acidconjugates of the compounds of the application.

Compounds of the application also include prodrugs or physiologicallyequivalent derivatives. A “prodrug” or “physiologically equivalentderivative” includes a precursor form of the drug which is metabolicallyconverted in vivo to produce the active drug. The application furthercontemplates the use of prodrugs which are converted in vivo to the TGR5modulating compounds used in the methods of the application (see, e.g.,R. B. Silverman, 1992, The Organic Chemistry of Drug Design and DrugAction, Academic Press, Chap. 8). Such prodrugs can be used to alter thebiodistribution (e.g., to allow compounds which would not typicallycross the blood-brain barrier to cross the blood-brain barrier) or thepharmacokinetics of the TGR5 modulating compound. For example, ananionic group, e.g., a carboxylate, sulfate or sulfonate, can beesterified, e.g., with an alkyl group (e.g., a methyl group) or a phenylgroup, to yield an ester. When the ester is administered to a subject,the ester is cleaved, enzymatically or non-enzymatically, reductively orhydrolytically, to reveal the anionic group. Such an ester can becyclic, e.g., a cyclic sulfate or sulfone, or two or more anionicmoieties may be esterified through a linking group. An anionic group canbe esterified with moieties (e.g., acyloxymethyl esters) which arecleaved to reveal an intermediate TGR5 modulating compound whichsubsequently decomposes to yield the active TGR5 modulating compound. Inone embodiment, the prodrug is a reduced form of a carboxylate, sulfateor sulfonate, e.g., an alcohol or thiol, which is oxidized in vivo tothe TGR5 modulating compound. Furthermore, an anionic moiety can beesterified to a group which is actively transported in vivo, or which isselectively taken up by target organs.

The term “compounds of the application” refers to compounds having theformulae described herein.

The term “TGR5 modulator” means any compound that interacts with theTGR5 receptor. The interaction is not limited to a compound acting as anantagonist, agonist, partial agonist, or inverse agonist of the TGR5receptor. In one aspect, the compounds of the present application act asan antagonist of the TGR5 receptor. In another aspect, the compounds ofthe present application act as an agonist of the TGR5 receptor. Inanother aspect, the compounds of the present application act as apartial agonist of the TGR5 receptor. In another aspect, the compoundsof the present application act as an inverse agonist of the TGR5receptor.

The profile of a ligand, traditionally, endogenous or synthetic, ischaracterized by its intrinsic efficacy ‘e’ originally described byFurchgott in 1966. It is used to express the degree to which thedifferent ligands produce varying biological responses while occupyingthe same number of receptors. Generally, the term “agonist” means acompound that enhances the activity of another molecule or receptorsite. An agonist, by classical definition, whether an orthosteric,allosteric, inverse or a co-agonist has a property to bind to thereceptor, alter its receptor state and result in a biological action.Consequently, agonism is defined as a property of an agonist or a ligandto produce a biological action. In contrast, an “antagonist” isessentially an agonist with high affinity to the same receptormacromolecule, but with very less or negligible intrinsic efficacy, andthus sterically prevents the biological actions of an agonist. As aproperty, antagonism may be functional or physiological, where anagonist has a direct competition for the receptor site in former andopposing effects via a different receptor-messenger system in the later.More specifically, a TGR5 agonist is a receptor ligand or compound thatbinds to TGR5 and increases the concentration of cyclic adenosinemonophosphate (cAMP) by at least 20% in cells expressing the receptor.Conversely, a TGR5 antagonist would be a compound that antagonizes orblocks the activity of an agonist, thereby effecting a reduction in theconcentration of cAMP.

The present application relates to compounds having TGR5 receptormodulating activity and their use to treat and/or prevent variousdiseases including metabolic disease, inflammatory disease, autoimmunedisease, cardiac disease, kidney disease, cancer, and gastrolintestinaldisease. Further, the present application relates to compounds of theformulae described herein.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, polymers and othermaterials and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” is art-recognized, andincludes, for example, pharmaceutically acceptable materials,compositions or vehicles, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting any subject composition from one organ, or portion of thebody, to another organ, or portion of the body. Each carrier must be“acceptable” in the sense of being compatible with the other ingredientsof a subject composition and not injurious to the patient. In certainembodiments, a pharmaceutically acceptable carrier is non-pyrogenic.Some examples of materials which may serve as pharmaceuticallyacceptable carriers include: (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,sunflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

A “composition” or “pharmaceutically acceptable composition” is aformulation containing a compound of the application or salt, solvate,ester, tautomer, amino acid conjugate, or metabolite thereof. In oneembodiment, the pharmaceutical composition is in bulk or in unit dosageform. The unit dosage form is any of a variety of forms, including, forexample, a capsule, an IV bag, a tablet, a single pump on an aerosolinhaler, or a vial. The quantity of active ingredient (e.g., aformulation of a compound of the application or salts thereof) in a unitdose of composition is an effective amount and is varied according tothe particular treatment involved. One skilled in the art willappreciate that it is sometimes necessary to make routine variations tothe dosage depending on the age and condition of the patient. The dosagewill also depend on the route of administration. A variety of routes arecontemplated, including oral, ocular, ophthalmic, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, intranasal, and the like. Dosage forms for the topicalor transdermal administration of a compound of this application includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In another embodiment, the active compound ismixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that arerequired.

The term “treating”, as used herein, means relieving, lessening,reducing, eliminating, modulating, or ameliorating, i.e., causingregression of the disease state or condition.

The term “preventing”, as used herein means, to completely or almostcompletely stop a disease state or condition, from occurring in apatient or subject, especially when the patient or subject ispredisposed to such or at risk of contracting a disease state orcondition. Preventing can also include inhibiting, i.e., arresting thedevelopment, of a disease state or condition, and relieving orameliorating, i.e., causing regression of the disease state orcondition, for example when the disease state or condition may alreadybe present.

The term “reducing the risk of”, as used herein, means to lower thelikelihood or probability of a central nervous system disease,inflammatory disease and/or metabolic disease from occurring in apatient, especially when the patient or subject is predisposed to suchoccurrence.

“Combination therapy” (or “co-therapy”) includes the administration of acompound of the application and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents (i.e., the compound ofthe application and at least a second agent). The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present application. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to thesubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, oral routes, intravenous routes, intramuscularroutes, and direct absorption through mucous membrane tissues. Thetherapeutic agents can be administered by the same route or by differentroutes. For example, a first therapeutic agent of the combinationselected may be administered by intravenous injection while the othertherapeutic agents of the combination may be administered orally.Alternatively, for example, all therapeutic agents may be administeredorally or all therapeutic agents may be administered by intravenousinjection. The sequence in which the therapeutic agents are administeredis not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery ormechanical treatments). Where the combination therapy further comprisesa non-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

A “therapeutically effective amount” of a compound of the application,or a combination of compounds is an amount (quantity or concentration)of compound or compounds. In one embodiment, when a therapeuticallyeffective amount of a compound is administered to a subject in need oftreatment symptoms arising from the disease are ameliorated immediatelyor after administration of the compound one or more times. The amount ofthe compound to be administered to a subject will depend on theparticular disorder, the mode of administration, co-administeredcompounds, if any, and the characteristics of the subject, such asgeneral health, other diseases, age, sex, genotype, body weight andtolerance to drugs. The skilled artisan will be able to determineappropriate dosages depending on these and other factors.

The term “prophylactically effective amount” means an amount (quantityor concentration) of a compound of the present application, or acombination of compounds, that is administered to prevent or reduce therisk of a disease—in other words, an amount needed to provide apreventative or prophylactic effect. The amount of the present compoundto be administered to a subject will depend on the particular disorder,the mode of administration, co-administered compounds, if any, and thecharacteristics of the subject, such as general health, other diseases,age, sex, genotype, body weight and tolerance to drugs. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors.

The term “flash dose” refers to compound formulations that are rapidlydispersing dosage forms.

A “subject” includes mammals, e.g., humans, companion animals (e.g.,dogs, cats, birds, and the like), farm animals (e.g., cows, sheep, pigs,horses, fowl, and the like) and laboratory animals (e.g., rats, mice,guinea pigs, birds, and the like). Typically, the subject is human.

Compounds and Compositions

In one aspect, the application relates to a compound of formula A:

or a pharmaceutically acceptable salt, solvate, ester, tautomer, aminoacid conjugate, or metabolite thereof, wherein:

A is

oxadiazolonyl, or isoxazolonyl, wherein the carbon atom marked with “*”is bonded to the carbon atom to which A is bonded;

n is 0, 1, or 2;

R₁ is H or OH;

R₂ is H or OH;

R₃ is CR₁₁R₁₂C(O)OH, C(O)NHR₃₁, tetrazolyl, oxadiazolyl, oxadiazolonyl,or thiazolidine-dionyl optionally substituted with NHS(O)₂—(C₁-C₃)alkyl;

R₁₁ and R₁₂ are each independently H, F, OH, CH₂OH, or CH₂F, providedthat R₁₁ and R₁₂ are not both H;

R₃₁ is OH, (CH₂)_(p)OH, or (CH₂)_(p)OSO₃H; and

p is 1 or 2.

(1) For example, n is 0.

(2) For example, n is 1 or 2. For example, n is 1. For example, n is 2.

(3) For example, R₁ and R₂ are each H.

(4) For example, R₁ is H, and R₂ is OH.

(5) For example, R₂ is H, and R₁ is OH.

(6) For example, R₂ is H, and R₁ is α-OH.

(7) For example, R₂ is H, and R₁ is β-OH.

(8) For example, R₁ and R₂ are each OH.

(9) For example, R₁ is α-OH, and R₂ is OH.

(10) For example, R₁ is β-OH, and R₂ is OH.

(11) For example, R₃ is CR₁₁R₁₂C(O)OH.

-   -   (I1) For example, R₁₁ is H, and R₁₂ is F, OH, CH₂OH, or CH₂F.    -   (I2) For example, R₁₁ is H, and R₁₂ is α-F, α-OH, α-CH₂OH, or        α-CH₂F.    -   (I3) For example, R₁₁ is H, and R₁₂ is β-F, β-OH, β-CH₂OH, or        β-CH₂F.    -   (I4) For example, R₁₁ is F, and R₁₂ is F, CH₂OH, or CH₂F.    -   (I5) For example, R₁₁ is F, and R₁₂ is F.    -   (I6) For example, R₁₁ is F, and R₁₂ is CH₂OH or CH₂F.    -   (I7) For example, R₁₁ is F, and R₁₂ is α-CH₂OH or α-CH₂F.    -   (I8) For example, R₁₁ is F, and R₁₂ is β-CH₂OH or β-CH₂F.    -   (I9) For example, R₁₁ is OH, and R₁₂ is CH₂OH or CH₂F.    -   (I10) For example, R₁₁ is OH, and R₁₂ is α-CH₂OH or α-CH₂F.    -   (I11) For example, R₁₁ is OH, and R₁₂ is β-CH₂OH or β-CH₂F.    -   (I12) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂OH or CH₂F.    -   (I13) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂OH.    -   (I14) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂F.    -   (I15) For example, R₁₁ is CH₂OH, and R₁₂ is α-CH₂F.    -   (I16) For example, R₁₁ is CH₂OH, and R₁₂ is β-CH₂F.    -   (I17) For example, R₁₁ is CH₂F, and R₁₂ is CH₂F.    -   (I2) For example, R₃ is tetrazolyl, oxadiazolyl, oxadiazolonyl,        or thiazolidine-dionyl optionally substituted with        NHS(O)₂—(C₁-C₃)alkyl. For example, R₃ is tetrazolyl,        1,3,4-oxadiazolyl, 1,2,4-oxadiazolonyl, or        thiazolidine-2,4-dionyl substituted with NHS(O)₂CH₃.    -   (I3) For example, R₃ is C(O)NHR₃₁.        -   (III1) For example, R₃₁ is OH.        -   (III2) For example, R₃₁ is (CH₂)_(p)OH.        -   (III3) For example, R₃₁ is (CH₂)₂OH        -   (III4) For example, R₃₁ is (CH₂)_(p)OSO₃H.        -   (III5) For example, R₃₁ is (CH₂)₂OSO₃H.        -   (III6) For example, p is 1.        -   (III7) For example, p is 2.

(14) For example, A is

(15) For example, A is oxadiazolonyl or isoxazolonyl. For example, A is1,2,4-oxadiazolonyl or isoxazolonyl.

For example, each of the substituents defined for one of A, n, p, R₁,R₂, R₃, R₁₁, R₁₂, and R₃₁, can be combined with any of the substituentsdefined for the others of A, n, p, R₁, R₂, R₃, R₁₁, R₁₂, and R₃₁.

(16) For example, n is 0, R₃ is CR₁₁R₁₂C(O)OH, and R₁₁ and R₁₂ are eachas defined in any of (I1)-(I17).

(17) For example, n is 1, R₃ is CR₁₁R₁₂C(O)OH, and R₁₁ and R₁₂ are eachas defined in any of (I1)-(I17).

(18) For example, n is 1 or 2, and R₃ is a tetrazolyl, oxadiazolyl,oxadiazolonyl, or thiazolidine-dionyl optionally substituted withNHS(O)₂—(C₁-C₃)alkyl.

(19) For example, n is 0, and R₃ is oxadiazolonyl.

(20) For example, n is 1, R₃ is C(O)NHR₃₁, and R₃₁ is as defined in anyof (III1)-(III5).

For example, the compound of formula A is a compound of formula I:

or a pharmaceutically acceptable salt, solvate, ester, tautomer, aminoacid conjugate, or metabolite thereof, wherein:

R₁₁ and R₁₂ are each independently H, F, OH, CH₂OH, or CH₂F, providedthat R₁₁ and R₁₂ are not both H; and

R₁₃ is H or OH.

(I1) For example, R₁₁ is H, and R₁₂ is F, OH, CH₂OH, or CH₂F.

(I2) For example, R₁₁ is H, and R₁₂ is α-F, α-OH, α-CH₂OH, or α-CH₂F.

(I3) For example, R₁₁ is H, and R₁₂ is β-F, β-OH, β-CH₂OH, or β-CH₂F.

(I4) For example, R₁₁ is F, and R₁₂ is F, CH₂OH, or CH₂F.

(I5) For example, R₁₁ is F, and R₁₂ is F.

(I6) For example, R₁₁ is F, and R₁₂ is CH₂OH or CH₂F.

(I7) For example, R₁₁ is F, and R₁₂ is α-CH₂OH or α-CH₂F.

(I8) For example, R₁₁ is F, and R₁₂ is β-CH₂OH or β-CH₂F.

(I9) For example, R₁₁ is OH, and R₁₂ is CH₂OH or CH₂F.

(I10) For example, R₁₁ is OH, and R₁₂ is α-CH₂OH or α-CH₂F.

(I11) For example, R₁₁ is OH, and R₁₂ is β-CH₂OH or β-CH₂F.

(I12) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂OH or CH₂F.

(I13) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂OH.

(I14) For example, R₁₁ is CH₂OH, and R₁₂ is CH₂F.

(I15) For example, R₁₁ is CH₂OH, and R₁₂ is α-CH₂F.

(I16) For example, R₁₁ is CH₂OH, and R₁₂ is β-CH₂F.

(I17) For example, R₁₁ is CH₂F, and R₁₂ is CH₂F.

(I18) For example, R₁₃ is H.

(I19) For example, R₁₃ is OH.

For example, each of the substituents defined for one of R₁₁, R₁₂, andR₁₃, can be combined with any of the substituents defined for the othertwo of R₁₁, R₁₂, and R₁₃.

(I20) For example, R₁₃ is H, and R₁₁ and R₁₂ are each as defined in anyof (I1)-(I17).

(I21) For example, R₁₃ is OH, and R₁₁ and R₁₂ are each as defined in anyof (I1)-(I17).

For example, the compound of formula A is a compound of formula II:

or a pharmaceutically acceptable salt, solvate, ester, tautomer, aminoacid conjugate, or metabolite thereof, wherein:

q is 0, 1, or 2;

R₂₁ and R₂₂ are each independently H or OH; and

R₂₃ is tetrazolyl, oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyloptionally substituted with NHS(O)₂—(C₁-C₃)alkyl.

(II1) For example, q is 0.

(II2) For example, q is 1.

(II3) For example, q is 2.

(II4) For example, R₂₁ and R₂₂ are each H.

(II5) For example, R₂₁ is H, and R₂₂ is OH.

(II6) For example, R₂₂ is H, and R₂₁ is OH.

(II7) For example, R₂₂ is H, and R₂₁ is α-OH.

(II8) For example, R₂₂ is H, and R₂₁ is β-OH.

(II9) For example, R₂₁ and R₂₂ are each OH.

(II10) For example, R₂₂ is OH, and R₂₁ is α-OH.

(II11) For example, R₂₂ is OH, and R₂₁ is β-OH.

(II12) For example, R₂₃ is tetrazolyl, oxadiazolyl, oxadiazolonyl, orthiazolidine-dionyl optionally substituted with NHS(O)₂—(C₁-C₃)alkyl.For example, R₂₃ is tetrazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolonyl,or thiazolidine-2,4-dionyl substituted with NHS(O)₂CH₃.

For example, each of the substituents defined for one of q, R₂₁, R₂₂,and R₂₃, can be combined with any of the substituents defined for theother three of q, R₂₁, R₂₂, and R₂₃.

(II13) For example, q is 0, and R₂₁ and R₂₂ are each as defined in anyof (II4)-(II11), and R₂₃ is as defined in (II12).

(II14) For example, q is 0, R₂₁ is H, R₂₂ is H, and R₂₃ isoxadiazolonyl.

(II15) For example, q is 0, R₂₁ is H, R₂₂ is OH, and R₂₃ isoxadiazolonyl.

(II16) For example, q is 1, and R₂₁ and R₂₂ are each as defined in anyof (II4)-(II11), and R₂₃ is as defined in (II12).

(II17) For example, q is 1, R₂₁ is H, R₂₂ is H, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

(II18) For example, q is 1, R₂₁ is H, R₂₂ is OH, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

(II19) For example, q is 2, and R₂₁ and R₂₂ are each as defined in anyof (II4)-(II11), and R₂₃ is as defined in (II12).

(II20) For example, q is 2, R₂₁ is H, R₂₂ is H, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

(II21) For example, q is 2, R₂₁ is OH, R₂₂ is H, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

(II22) For example, q is 2, R₂₁ is H, R₂₂ is OH, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

(II23) For example, q is 2, R₂₁ is OH, R₂₂ is OH, and R₂₃ is tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl.

For example, the compound of formula A is a compound of formula III:

or a pharmaceutically acceptable salt, solvate, ester, tautomer, aminoacid conjugate, or metabolite thereof, wherein:

R₃₁ is OH, (CH₂)_(p)OH, or (CH₂)_(p)OSO₃H; and

p is 1 or 2.

(I111) For example, p is 1.

(II12) For example, p is 2.

(II13) For example, R₃₁ is OH.

(II14) For example, R₃₁ is (CH₂)_(p)OH.

(II15) For example, R₃₁ is (CH₂)₂OH

(II16) For example, R₃₁ is (CH₂)_(p)OSO₃H.

(II17) For example, R₃₁ is (CH₂)₂OSO₃H.

For example, each of the substituents defined for one of p and R₃₁ canbe combined with any of the substituents defined for the other of p andR₃₁.

(III17) For example, p is 1, and R₃₁ is as defined in (II13)-(II17).

(III18) For example, p is 2, and R₃₁ is as defined in (II13)-(II17).

Representative compounds of the application are listed in Table 1.

TABLE 1 Cmpd No. Chemical Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

In one aspect, the application includes a compound of the application,wherein the compound is a pharmaceutically acceptable salt.

One aspect of the application includes a composition comprising acompound of the application or a pharmaceutically acceptable salt,solvate, ester, tautomer, amino acid conjugate, or metabolite thereof,and at least one pharmaceutically acceptable excipient.

Synthesis of the Compounds of the Application

The present application provides methods for the synthesis of thecompounds of each of the formulae described herein. The presentapplication also provides detailed methods for the synthesis of variousdisclosed compounds of the present application according to thefollowing schemes as shown in the examples.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where methods or processes are described ashaving, including, or comprising specific process steps, the processesalso consist essentially of, or consist of, the recited processingsteps. Further, it should be understood that the order of steps or orderfor performing certain actions is immaterial so long as the applicationremains operable. Moreover, two or more steps or actions can beconducted simultaneously.

The synthetic processes of the application can tolerate a wide varietyof functional groups, therefore various substituted starting materialscan be used. The processes generally provide the desired final compoundat or near the end of the overall process, although it may be desirablein certain instances to further convert the compound to apharmaceutically acceptable salt, ester or prodrug thereof.

Compounds of the present application can be prepared in a variety ofways using commercially available starting materials, compounds known inthe literature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or which will be apparent to the skilled artisan in light ofthe teachings herein. Standard synthetic methods and procedures for thepreparation of organic molecules and functional group transformationsand manipulations can be obtained from the relevant scientificliterature or from standard textbooks in the field. Although not limitedto any one or several sources, classic texts such as Smith, M. B.,March, J., March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure, 5th edition, John Wiley & Sons: New York, 2001; andGreene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis,3rd edition, John Wiley & Sons: New York, 1999, incorporated byreference herein, are useful and recognized reference textbooks oforganic synthesis known to those in the art. The following descriptionsof synthetic methods are designed to illustrate, but not to limit,general procedures for the preparation of compounds of the presentapplication.

Compounds of the present application can be conveniently prepared by avariety of methods familiar to those skilled in the art. The compoundseach of the formulae described herein may be prepared according to thefollowing procedures from commercially available starting materials orstarting materials which can be prepared using literature procedures.These procedures show the preparation of representative compounds ofthis application.

All the abbreviations used in this application are found in ProtectiveGroups in Organic Synthesis by John Wiley & Sons, Inc, or the MERCKINDEX by MERCK & Co., Inc, or other chemistry books or chemicalscatalogs by chemicals vendor such as Aldrich, or according to usage knowin the art.

Use and Methods

The application includes the use of a compound or a pharmaceuticallyacceptable salt, solvate, ester, tautomer, amino acid conjugate, ormetabolite, in the manufacture of a medicament for a treating orpreventing disease in a subject. The application also includes a methodof treating or preventing disease in a subject by administering to thesubject a compound of the application or a pharmaceutically acceptablesalt, solvate, ester, tautomer, amino acid conjugate, or metabolite.

One aspect of the application includes the use or method, wherein thedisease is a disease in which TGR5 is involved, i.e., a “TGR5-mediateddisease”. In one embodiment, the TGR5-mediated disease is selected frommetabolic disease, inflammatory disease, autoimmune disease, cardiacdisease, kidney disease, cancer, and gastrointestinal disease in whichTGR5 is involved.

In one aspect, the metabolic disease is selected from obesity, diabetes(and complications arising from diabetes, such as diabetic nephropathy,diabetic neuropathy, diabetic retinopathy, etc.), diabesity, metabolicsyndrome, insulin resistance, including pre-diabetic insulin resistance,hypertension, and dyslipidemia. In one aspect, the metabolic disease isobesity. In another aspect, the metabolic disease is diabetes. In oneaspect, diabetes is selected from pre-diabetes and type II diabetes. Inone aspect, the metabolic disease is metabolic syndrome. In one aspect,the metabolic disease is insulin resistance. In one aspect, themetabolic disease is dyslipidemia. In one aspect, the metabolic diseaseis diabesity. The term “diabesity” refers to a condition wherein thesubject has both diabetes and excessive weight.

In one aspect, the inflammatory disease is selected from allergy,osteoarthritis (OA), chronic obstructive pulmonary disease (COPD),appendicitis, bronchial asthma, pancreatitis, allergic rash, andpsoriasis.

In one aspect, the autoimmune disease is selected from rheumatoidarthritis, multiple sclerosis, and type I diabetes. In one aspect, theautoimmune disease is erythematosus.

In one aspect, the cardiac disease is selected from congestive heartfailure, myocardial infarction, atherosclerosis, angina pectoris,arteriosclerosis and cerebrovascular disease (hemorrhage, stroke,cerebrovascular infarction).

In one aspect, the kidney disease is selected from diabetic nephropathy,chronic renal failure, glomerular nephritis, hypertensivenephrosclerosis, chronic glomerulonephritis, chronic transplantglomerulopathy, chronic interstitial nephritis, and polysystic kidneydisease.

In one aspect, the gastrointestinal disease is selected frominflammatory bowel disease (Crohn's disease, ulcerative colitis), shortbowel syndrome (post-radiation colitis), microscopic colitis, irritablebowel syndrome (malabsorption), and bacterial overgrowth.

In one aspect, the cancer is selected from colorectal cancer, livercancer, hepatocellular carcinoma, cholangio carcinoma, renal cancer,gastric cancer, pancreatic cancer, prostate cancer, and insulanoma.

In one aspect, the application includes a use or method, wherein thecompound of the application is a TGR5 agonist.

In one aspect, the application includes a use or method, wherein thecompound or composition is administered to the subject orally, ocularly,ophthalmically, parentally, intravenously, or topically. In one aspect,the subject is human.

The application includes a use or method comprising administering to asubject a therapeutically effective amount of the compound of theapplication. The application also includes a use or method comprisingadministering to a subject a prophylatically effective amount of thecompound of the application.

The compounds and compositions of the present application can beadministered by various routes, e.g., oral, subcutaneous, intramuscular,intravenous, or intraperitoneal. The preferred routes of administrationare oral, subcutaneous, and intravenous at daily doses of about0.01-5000 mg, preferably 5-500 mg, of the compound of the applicationfor a 70 kg adult human per day. The appropriate dose may beadministered in a single daily dose or as divided doses presented atappropriate intervals, for example as two, three, four, or more subdosesper day.

For preparing pharmaceutical compositions containing a compound of theapplication, inert and pharmaceutically acceptable carriers are used.The pharmaceutical carrier can be either solid or liquid. Solid formpreparations include, for example, powders, tablets, dispersiblegranules, capsules, cachets, and suppositories. A solid carrier can beone or more substances that can also act as diluents, flavoring agents,solubilizers, lubricants, suspending agents, binders, or tabletdisintegrating agents; it can also be an encapsulating material.

In powders, the carrier is generally a finely divided solid that is in amixture with the finely divided active component, e.g., a compound ofthe application. In tablets, the active ingredient is mixed with thecarrier having the necessary binding properties in suitable proportionsand compacted in the shape and size desired.

For preparing pharmaceutical compositions in the form of suppositories,a low-melting wax such as a mixture of fatty acid glycerides and cocoabutter is first melted and the active ingredient is dispersed thereinby, for example, stirring. The molten homogeneous mixture is then pouredinto convenient-sized molds and allowed to cool and solidify.

Powders and tablets preferably contain between about 5% to about 70% byweight of the active ingredient of the compound of the application.Suitable carriers include, for example, magnesium carbonate, magnesiumstearate, talc, lactose, sugar, pectin, dextrin, starch, tragacanth,methyl cellulose, sodium carboxymethyl cellulose, a low-melting wax,cocoa butter, and the like.

The pharmaceutical compositions can include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the compound of the application (with or without othercarriers) is surrounded by the carrier, such that the carrier is inassociation with the compound. In a similar manner, cachets can also beincluded. Tablets, powders, cachets, and capsules can be used as soliddosage forms suitable for oral administration.

Liquid pharmaceutical compositions include, for example, solutionssuitable for oral, ocular, ophthalmic, or parenteral administration,suspensions, and emulsions suitable for oral administration. Sterilewater solutions of the active component or sterile solutions of theactive component in solvent comprising water, buffered water, saline,PBS, ethanol, or propylene glycol are examples of liquid compositionssuitable for parenteral administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, detergents, and thelike.

Sterile solutions can be prepared by dissolving the active component(e.g., a compound of the application) in the desired solvent system, andthen passing the resulting solution through a membrane filter tosterilize it or, alternatively, by dissolving the sterile compound in apreviously sterilized solvent under sterile conditions. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile aqueous carrierprior to administration. The pH of the preparations typically will bebetween 3 and 11, more preferably from 5 to 9, and most preferably from7 and 8.

The pharmaceutical compositions containing compounds of the applicationcan be administered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions are administered in an amountsufficient to cure, reverse, or at least partially slow or arrest thesymptoms of the disease and its complications. An amount adequate tocure, reverse, or at least partially slow or arrest the symptom of thedisease and its complications is defined as a “therapeutically effectivedose”. In prophylatic applications, compositions are administered in anamount sufficient to prevent the symptoms of the disease and itscomplications. An amount adequate to prevent the symptom of the diseaseand its complications is defined as a “prophylatically effective dose”.

Amounts effective for therapeutic use will depend on the severity of thedisease or condition and the weight and general state of the patient,but generally range from about 0.1 mg to about 2,000 mg of the compoundper day for a 70 kg patient, with dosages of from about 5 mg to about500 mg of the compound per day for a 70 kg patient being more commonlyused.

In prophylactic applications, pharmaceutical compositions containingcompounds of the application are administered to a patient susceptibleto or otherwise at risk of developing disease, in an amount sufficientto delay or prevent the onset of the disease symptoms. In this use, theprecise amounts of the compound again depend on the patient's state ofhealth and weight, but generally range from about 0.1 mg to about 2,000mg for a 70 kg patient per day, more commonly from about 5 mg to about500 mg for a 70 kg patient per day.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of a compound of the application sufficient to effectivelytreat or prevent disease in the patient.

The application also provides kits for preventing or treating diseaseaccording to the use and method of the present application. In oneaspect, the application includes kit for treating or preventing diseasein a subject, wherein the kit comprises a compound of the application ora salt, solvate, ester, tautomer, amino acid conjugate, or metabolitethereof.

The kits typically include a pharmaceutical composition that contains aneffective amount of a compound of the application, as well asinformational material containing instructions of how to dispense thepharmaceutical composition, including description of the type ofpatients who may be treated, the schedule (e.g., dose and frequency) androute of administration, and the like.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The applicationhaving now been described by way of written description, those of skillin the art will recognize that the application can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

EXAMPLES Example 1: Synthesis of Compound 1

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed withsaturated NaHCO₃ (200 mL), H₂O (200 mL) and brine (200 mL). The organiclayer was dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was then dissolved in CH₂Cl₂ (180 mL) and theresulting solution was treated with diisopropylethylamine (94 mL, 550.5mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol) andmethoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirred andrefluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

(E+Z)-3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-24,24-trimetylsilyloxy-methoxy-5β-chol-23-ene(3)

To a stirred solution of diisopropylamine (11.7 mL, 82.5 mmol) indistilled THF (40 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (32.0 mL, 79.3 mmol) was added dropwise. After 15′, thesolution was cooled up to −78° C. and chlorotrimethylsilane (12.7 mL,84.5 mmol) was added dropwise. After additional 15′, a solution of 3(6.0 g, 10.30 mmol) in distilled THF (20 mL) was added portionwise inabout 20′ maintaining the internal temperature not over −70° C. Once theaddition was completed, the reaction mixture was stirred at −78° C. for1 hr and then warmed at room temperature. Volatiles were removed underreduced pressure, and the residue was suspended in petroleum ether (80mL) and filtered under vacuum. The liquor was concentrated under reducedpressure, to give 10.12 g of oil residue that was used for the next stepwithout further purification.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(S)-hydroxy-5β-cholan-24-oate(4)

To a suspension of freshly crystallized and acetic acid freelead(IV)tetraacetate (6.85 g, 15.46 mmol) in distilled CH₂Cl₂ (50 mL)under N₂ atmosphere, a solution of 3 (10.12 g) in CH₂Cl₂ (30 mL) wasadded dropwise. After 30′ the reaction mixture was filtered under vacuumthrough a celite pad. The filtrate was concentrated under reducedpressure and the residue was filtered through a silica gel pad (h: 6 cm,φ: 2 cm) collecting the crude reaction mixture with petroleumether/AcOEt (8:2, v/v). After solvent evaporation, the residue (6.50 g)was dissolved in MeOH (50 mL) and treated with potassium carbonate (2.13g, 15.5 mmol) at room temperature for 15′. The mixture was then dilutedwith CH₂Cl₂ (50 mL) and filtered under vacuum. The filtrate was furtherdiluted with CH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueousphase was extracted with CH₂Cl₂ (3×40 mL), and the collected organiclayers were dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was purified by medium pressure liquidchromatography and collecting the desired compound with an isocraticelution constituted by petroleum ether/AcOEt (65:35, v/v). in 19% yield.

¹H-NMR (CDCl₃, 200 MHz) δ 0.65 (3H, s, 18-CH₃), 0.81-0.88 (6H, m,19-CH₃+CH₂CH₃), 0.99 (3H, d, J=6.4 Hz, 21-CH₃), 3.32-3.36 (1H, m, 3-CH),3.33 (6H, m, 2×OCH₂OCH₃), 3.39 (3H, s, OCH₂OCH₃), 3.46 (1H, s, 7-CH),3.74 (3H, s, CO₂CH₃), 3.76 (1H, s, 12-CH), 4.18 (1H, t, J=6.6 Hz,23-CH), 4.51-4.72 (6H, m, 3×OCH₂OCH₃). ¹³C-NMR (CDCl₃, 50.3 MHz) δ 11.7,12.4, 18.7, 22.8, 23.0, 23.8, 24.9, 27.3, 27.6, 27.9, 30.3, 33.5, 35.5(×2), 40.7, 41.2, 41.8, 42.2, 45.8, 46.3, 46.7, 52.2, 54.9, 55.7, 55.9,69.9, 77.4, 79.9, 80.0, 94.3, 95.8, 98.4, 176.0.

3α,7α,12α,23(S)-tetrahydroxy-6α-ethyl-5β-cholan-24-oic acid (Compound 1)

To a solution of 4 (0.10 g, 0.17 mmol) in MeOH (7 mL), HCl 3 N (0.60 mL,1.80 mmol) was added, and the mixture was stirred at 45° C. for 18 hrs.Sodium hydroxide (0.10 g, 2.50 mmol) was added and the mixture wasstirred at 45° C. for additional 5 hrs. MeOH was removed under reducedpressure, the residue was diluted with H₂O up to 10 mL and washed withEt₂O (2×5 mL). The aqueous phase was acidified with HCl 3 N, extractedwith CH₃Cl₃/MeOH (85:15, v/v) (5×10 mL) and concentrated under reducedpressure. The resulting residue was purified by RP-18 medium pressureliquid chromatography by using H₂O/MeOH (9:1→1:1, v/v) as eluent toobtain the desired compound Compound 1 in 78% yield.

rf: 0.11 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 60:40). ¹H-NMR(D20, 400 MHz) δ 0.58 (3H, s, 18-CH₃), 0.72-0.75 (6H, m, 19-CH₃+CH₂CH₃),0.90 (3H, d, J=6.0 Hz, 21-CH₃), 3.25-3.34 (1H, m, 3-CH), 3.62 (1H, s,7-CH), 3.89 (1H, t, J=8.0 Hz, 23-CH), 3.93 (1H, s, 12-CH). ¹³C-NMR (D20,100.6 MHz) δ 11.0, 11.9, 17.8, 21.8, 22.4, 22.8, 26.7, 27.4, 27.7, 29.1,32.4, 33.3, 34.7, 39.7, 41.0, 41.1, 41.6, 44.8, 46.2, 47.3, 48.8, 70.4,71.9 (×2), 73.1, 182.0.

Example 2: Synthesis of Compound 2

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

(E+Z)-3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-24,24-trimetylsilyloxy-methoxy-5β-chol-23-ene(3)

To a stirred solution of diisopropylamine (11.7 mL, 82.5 mmol) indistilled THF (40 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (32.0 mL, 79.3 mmol) was added dropwise. After 15′, thesolution was cooled up to −78° C. and chlorotrimethylsilane (12.7 mL,84.5 mmol) was added dropwise. After additional 15′, a solution of 3(6.0 g, 10.30 mmol) in distilled THF (20 mL) was added portionwise inabout 20′ maintaining the internal temperature not over −70° C. Once theaddition was completed, the reaction mixture was stirred at −78° C. for1 hr and then warmed at room temperature. Volatiles were removed underreduced pressure, and the residue was suspended in petroleum ether (80mL) and filtered under vacuum. The liquor was concentrated under reducedpressure, to give 10.12 g of oil residue that was used for the next stepwithout further purification.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(R)-hydroxy-5β-cholan-24-oate(4)

To a suspension of freshly crystallized and acetic acid freelead(IV)tetraacetate (6.85 g, 15.46 mmol) in distilled CH₂Cl₂ (50 mL)under N₂ atmosphere, a solution of 3 (10.12 g) in CH₂Cl₂ (30 mL) wasadded dropwise. After 30′ the reaction mixture was filtered under vacuumthrough a celite pad. The filtrate was concentrated under reducedpressure and the residue was filtered through a silica gel pad (h: 6 cm,φ: 2 cm) collecting the crude reaction mixture with petroleumether/AcOEt (8:2, v/v). After solvent evaporation, the residue (6.50 g)was dissolved in MeOH (50 mL) and treated with potassium carbonate (2.13g, 15.5 mmol) at room temperature for 15′. The mixture was then dilutedwith CH₂Cl₂ (50 mL) and filtered under vacuum. The filtrate was furtherdiluted with CH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueousphase was extracted with CH₂Cl₂ (3×40 mL), and the collected organiclayers were dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was purified by medium pressure liquidchromatography and collecting the desired compound with an isocraticelution constituted by petroleum ether/AcOEt (65:35, v/v), to obtain 4in 20% yield.

¹H-NMR (CDCl₃, 200 MHz) δ 0.69 (3H, s, 18-CH₃), 0.83-0.90 (6H, m,19-CH₃+CH₂CH₃), 1.01 (3H, d, J=6.3 Hz, 21-CH₃), 3.24-3.35 (1H, m, 3-CH),3.34 (6H, m, 2×OCH₂OCH₃), 3.42 (3H, s, OCH₂OCH₃), 3.48 (1H, s, 7-CH),3.76 (3H, s, CO₂CH₃), 3.81 (1H, s, 12-CH), 4.20 (1H, dd, J₁=1.9 Hz,J₂=6.0 Hz 23-CH), 4.56-4.74 (6H, m, 3×OCH₂OCH₃). ¹³C-NMR (CDCl₃, 50.3MHz) δ 11.8, 12.5, 17.2, 28.8, 23.0, 23.8, 24.9, 27.4, 27.6, 27.8, 30.3,32.4, 35.5 (×2), 40.7, 41.0, 41.9, 42.3, 45.8, 46.4, 46.5, 52.4, 54.9,55.7, 55.9, 68.0, 77.4, 80.0, 81.0, 94.3, 95.9, 98.4, 176.5.

3α,7α,12α,23(R)-tetrahydroxy-6α-ethyl-5β-cholan-24-oic acid (Compound 2)

To a solution of 4a or 4b (0.10 g, 0.17 mmol) in MeOH (7 mL), HCl 3 N(0.60 mL, 1.80 mmol) was added, and the mixture was stirred at 45° C.for 18 hrs. Sodium hydroxide (0.10 g, 2.50 mmol) was added and themixture was stirred at 45° C. for additional 5 hrs. MeOH was removedunder reduced pressure, the residue was diluted with H₂O up to 10 mL andwashed with Et₂O (2×5 mL). The aqueous phase was acidified with HCl 3 N,extracted with CH₃Cl₃/MeOH (85:15, v/v) (5×10 mL) and concentrated underreduced pressure. The resulting residue was purified by RP-18 mediumpressure liquid chromatography by using H₂O/MeOH (9:1→1:1, v/v) aseluent to obtain the desired compound Compound 2 in 71% yield.

rf: 0.10 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 60:40). ¹H-NMR(D20, 400 MHz) δ 0.66 (3H, s, 18-CH₃), 0.78-0.86 (6H, m, 19-CH₃+CH₂CH₃),0.96 (3H, pss, 21-CH₃), 1.96-2.00 (1H, m, 22-CH₂), 3.30-3.37 (1H, m,3-CH), 3.66 (1H, s, 7-CH), 3.95 (1H, m, 23-CH), 4.01 (1H, s, 12-CH).¹³C-NMR (D20, 100.6 MHz) δ 11.2, 12.2, 16.1, 21.9, 22.6, 22.8, 26.8,27.3, 27.9, 29.1, 32.4, 34.8, 35.2, 39.9, 40.8, 41.3, 41.7, 45.0, 46.3(×2), 47.3, 69.9, 70.4, 71.8, 73.0, 182.7.

Example 3: Synthesis of Compound 3

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

(E+Z)-3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-24,24-trimetylsilyloxy-methoxy-5β-chol-23-ene(3)

To a stirred solution of diisopropylamine (11.7 mL, 82.5 mmol) indistilled THF (40 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (32.0 mL, 79.3 mmol) was added dropwise. After 15′, thesolution was cooled up to −78° C. and chlorotrimethylsilane (12.7 mL,84.5 mmol) was added dropwise. After additional 15′, a solution of 3(6.0 g, 10.30 mmol) in distilled THF (20 mL) was added portionwise inabout 20′ maintaining the internal temperature not over −70° C. Once theaddition was completed, the reaction mixture was stirred at −78° C. for1 hr and then warmed at room temperature. Volatiles were removed underreduced pressure, and the residue was suspended in petroleum ether (80mL) and filtered under vacuum. The liquor was concentrated under reducedpressure, to give 10.12 g of oil residue that was used for the next stepwithout further purification.

Methyl23(R+S)-hydroxy-6α-ethyl-3α,7α,12α-trimethoxymethyloxy-5β-cholan-24-oate(4)

To a suspension of freshly crystallized and acetic acid freelead(IV)tetraacetate (6.85 g, 15.46 mmol) in distilled CH₂Cl₂ (50 mL)under N₂ atmosphere, a solution of 3 (10.12 g) in CH₂Cl₂ (30 mL) wasadded dropwise. After 30′ the reaction mixture was filtered under vacuumthrough a celite pad. The filtrate was concentrated under reducedpressure and the residue was filtered through a silica gel pad (h: 6 cm,φ: 2 cm) collecting the crude reaction mixture with petroleumether/AcOEt (8:2, v/v). After solvent evaporation, the residue (6.50 g)was dissolved in MeOH (50 mL) and treated with potassium carbonate (2.13g, 15.5 mmol) at room temperature for 15′. The mixture was then dilutedwith CH₂Cl₂ (50 mL) and filtered under vacuum. The filtrate was furtherdiluted with CH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueousphase was extracted with CH₂Cl₂ (3×40 mL), and the collected organiclayers were dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography byusing petroleum ether/AcOEt (9:1→7:3, v/v) as eluent to afford 2.53 g(4.26 mmol, 41%) of 4 as mixture of two epimers.

Methyl 23-oxo-6α-ethyl-3α,7α,12α-trimethoxymethyloxy-5β-cholan-24-oate(5)

To a solution of oxalyl chloride (4.0 mL, 46.7 mmol) in distilled CH₂Cl₂(70 mL) under N₂ atmosphere and cooled ad −60° C., DMSO (6.60 mL, 93.4mmol) diluted in CH₂Cl₂ (10 mL) was added dropwise. After 15′, asolution of 4 (11.2 g, 18.7 mmol) in CH₂Cl₂ (70 mL) was added dropwise,and the resulting mixture was stirred at −60° C. for 1 hr. Triethylamine(26.2 mL, 186.8 mmol) was added dropwise and the mixture was slowlywarmed at room temperature. The reaction mixture was treated with KOH 1M (100 mL) for 5′ and water and organic phases were separated. Theaqueous phase was then extracted with CH₂Cl₂ (2×50 mL). The collectedorganic layers were dried over Na₂SO₄ anhydrous and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography yielding pure intermediate 5 (6.82 g, 11.4 mmol, 61%)using a solution of petroleum ether/AcOEt (85:15, v/v).

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23,23-gemdifluoro-5β-cholan-24-oate(6)

To a solution of 5 (6.82 g, 11.4 mmol) in distilled CH₂Cl₂ (100 mL)under N₂ atmosphere, diethylaminosulfurtrifluoride (15.1 mL, 114.4 mmol)was added, and the reaction was stirred at room temperature for 8 hrs.The mixture was cautiously poured in a saturated solution of NaHCO₃ (250mL) placed in a water-ice bath and under magnetic stirring. Once the CO₂release was completed, the two phases were separated and the organiclayer was washed with H₂O (100 mL), brine (100 mL), dried over Na₂SO₄anhydrous and concentrated under reduced pressure. The residue waspurified by silica gel flash chromatography eluting with a solution ofpetroleum ether/AcOEt (9:1, v/v) to collect the desired compound 6 (5.17g, 8.4 mmol, 73%).

3α,7α,12α-Trihydroxy-6α-ethyl-23,23-gemdifluoro-5β-cholan-24-oic acid(Compound 3)

To a solution of 6 (5.17 g, 8.4 mmol) in MeOH (50 mL), HCl 3 N (25.1 mL,75.4 mmol) was added and the mixture was stirred at 45° C. for 18 hrs.Sodium hydroxide (5.0 g, 125.6 mmol) was added and the mixture reactedat 45° C. for additional 5 hrs. MeOH was then removed under reducedpressure and the residue was diluted with H₂O up to 70 mL and washedwith Et₂O (2×30 mL). The aqueous phase was acidified with HCl 3 N andthe resulting whitish suspension was filtered through a RP-18 silica gelpad (h: 4 cm, φ: 2 cm) under vacuum, washing with H₂O (250 mL) andcollecting the crude compound with H₂O/MeCN (1:1, v/v). Once the solventwas removed under reduced pressure, the residue was purified by RP-18medium pressure liquid chromatography by using H₂O/MeCN as eluent(8:2→6:4, v/v) to afford 3.59 g of pure Compound 3 (91%).

rf: 0.65 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(DMSO-d6, 400 MHz) δ 0.61 (3H, s, 18-CH₃), 0.80-0.86 (6H, m,19-CH₃+CH₂CH₃), 0.93 (3H, d, J=6.3 Hz, 21-CH₃), 3.30-3.36 (1H, m, 3-CH),3.48 (1H, s, 7-CH), 3.78 (1H, s, 12-CH), 3.78 (1H, s, OH), 3.97 (1H, s,OH), 4.17-4.21 (1H, bs, OH). ¹³C-NMR (DMSO-d6, 100.6 MHz) δ 11.7, 12.1,18.4, 22.18, 22.6, 22.9, 26.5, 27.4, 28.6, 30.2, 30.6, 30.8, 33.4, 34.8,35.5, 41.2, 41.6, 45.4, 45.9, 46.4, 68.3, 70.6, 70.8, 117.2 (t,J_(C-F)=248.7 Hz), 165.5 (t, J_(C-F)=31.9 Hz). ¹⁹F-NMR (DMSO-d6, 376.5MHz) δ −102.2 (2F, m). MS-TIC (−) m/z: 471.3.

Example 4: Synthesis of Compound 4

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(A+B)-hydroxymethyl-5β-cholan-24-oate(4)

To a solution of diisopropylamine (0.87 g, 8.59 mmol) in dry THF (25 mL)at −78° C., nBuLi 2.5 M in hexane (3.1 mL, 7.73 mmol) was addeddropwise. After 15′, a solution of compound 2 (0.50 g, 0.86 mmol) in dryTHF (10 mL) was added dropwise and the mixture was reacted at −78° C.for 15′. Ethylformate (1.27 g, 17.18 mmol) was then added and reactedfor 1 hr prior the reaction was allowed to warm to room temperature. Thefraction mixture was poured into H₂O (50 mL) and extracted with EtOAc(3×50 mL). The combined organic layers were washed with brine (100 mL),dried over Na₂SO₄ anhydrous and evaporated under reduced pressure. Theintermediate 3 thus obtained was dissolved in MeOH (20 mL) and treatedat 0° C. with NaBH₄ for 30′. The reaction was quenched with H₂O (50 mL)and extracted with CH₂Cl₂ (3×50 mL). The combined organic layers werewashed with H₂O (100 mL), brine (100 mL), dried over Na₂SO₄ andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography (eluting with isopropanol in CHCl₃ from 2 to 7%,v/v) obtaining 0.29 g of compound 4 as epimeric mixture (0.48 mmol,56%).

3α,7α,12α-Trihydroxy-6α-ethyl-23(A)-hydroxymethyl-5β-cholan-24-oic acid(Compound 4)

To a solution of compound 4 (0.29 g, 0.48 mmol) in MeOH (15 mL) HCl 3 N(5 mL) was added and the resulting mixture was stirred at 50° C. for 48hrs. The mixture was allowed to cool at room temperature and treatedwith NaOH (5% in MeOH) up to pH 14 at 45° C. for 24 hrs. The solvent wasevaporated under reduced pressure, the crude was suspended in H₂O (30mL) and extracted with Et₂O (2×10 mL). The aqueous phase was acidifiedwith HCl 3 N and the precipitate was collected by filtration. The crudecompound was purified by medium pressure liquid chromatography using asolution of H₂O/MeOH (MeOH from 10 to 40%). The epimer Compound 4 wasobtained in 29% yield (0.065 g, 0.14 mmol).

rf: 0.39 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeOH 20:80). ¹H-NMR(CD₃OD, 400 MHz) δ: 0.71 (3H, s, 18-CH₃), 0.89-0.92 (6H, m,19-CH₃+CH₂CH₃), 1.06 (3H, d, J=5.7 Hz, 21-CH₃), 2.55-2.62 (1H, bs,23-CH), 3.29-3.34 (1H, m, 3-CH), 3.55-3.64 (1H, s, 7-CH), 3.64-3.67 (2H,bs, CH₂OH), 3.97 (1H, s, 12-CH). ¹³C-NMR (CD₃OD, 100.6 MHz) δ: 12.6,13.8, 18.6, 24.0 (×2), 24.8, 28.8, 29.4, 30.2, 31.6, 34.9, 36.3, 36.8,37.6, 37.8, 42.3, 43.7 (×2), 47.4, 48.2, 66.6, 71.7, 73.7, 74.6, 176.3.

Example 5: Synthesis of Compound 5

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(A+B)-hydroxymethyl-5β-cholan-24-oate(4)

To a solution of diisopropylamine (0.87 g, 8.59 mmol) in dry THF (25 mL)at −78° C., nBuLi 2.5 M in hexane (3.1 mL, 7.73 mmol) was addeddropwise. After 15′, a solution of compound 2 (0.50 g, 0.86 mmol) in dryTHF (10 mL) was added dropwise and the mixture was reacted at −78° C.for 15′. Ethylformate (1.27 g, 17.18 mmol) was then added and reactedfor 1 hr prior the reaction was allowed to warm to room temperature. Theraction mixture was poured into H₂O (50 mL) and extracted with EtOAc(3×50 mL). The combined organic layers were washed with brine (100 mL),dried over Na₂SO₄ anhydrous and evaporated under reduced pressure. Theintermediate 3 thus obtained was dissolved in MeOH (20 mL) and treatedat 0° C. with NaBH₄ for 30′. The reaction was quenched with H₂O (50 mL)and extracted with CH₂Cl₂ (3×50 mL). The combined organic layers werewashed with H₂O (100 mL), brine (100 mL), dried over Na₂SO₄ andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography (eluting with isopropanol in CHCl₃ from 2 to 7%,v/v) obtaining 0.29 g of compound 4 as epimeric mixture (0.48 mmol,56%).

3α,7α,12α-Trihydroxy-6α-ethyl-23(B)-hydroxymethyl-5β-cholan-24-oic acid(Compound 5)

To a solution of compound 4 (0.29 g, 0.48 mmol) in MeOH (15 mL) HCl 3 N(5 mL) was added and the resulting mixture was stirred at 50° C. for 48hrs. The mixture was allowed to cool at room temperature and treatedwith NaOH (5% in MeOH) up to pH 14 at 45° C. for 24 hrs. The solvent wasevaporated under reduced pressure, the crude was suspended in H₂O (30mL) and extracted with Et₂O (2×10 mL). The aqueous phase was acidifiedwith HCl 3 N and the precipitate was collected by filtration. The crudecompound was purified by medium pressure liquid chromatography using asolution of H₂O/MeOH (MeOH from 10 to 40%). The epimer Compound 5 wasobtained in 40% yield (0.09 g, 0.19 mmol).

rf: 0.36 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 20:80). ¹H-NMR(CD₃OD, 400 MHz) δ: 0.72 (3H, s, 18-CH₃), 0.89-0.92 (6H, m,19-CH₃+CH₂CH₃), 1.065 (3H, d, J=6.0 Hz, 21-CH₃), 2.39-2.46 (1H, bs,23-CH), 3.28-3.33 (1H, m, 3-CH), 3.61 (2H, m, CH₂OH), 3.66 (1H, s,7-CH), 3.98 (1H, s, 12-CH). ¹³C-NMR (CD₃OD, 100.6 MHz) δ: 12.0, 13.0,18.0, 23.5 (×2), 24.2, 28.2, 29.0, 29.6, 30.7, 31.1, 34.4, 35.8, 36.3,36.7, 37.0, 41.8, 43.1 (×2), 47.0, 47.7, 64.4, 71.2, 73.2, 74.1, 184.5.

Example 6: Synthesis of Compound 6

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(A+B)-hydroxymethyl-5β-cholan-24-oate(4)

To a solution of diisopropylamine (0.87 g, 8.59 mmol) in dry THF (25 mL)at −78° C., nBuLi 2.5 M in hexane (3.1 mL, 7.73 mmol) was addeddropwise. After 15′, a solution of compound 2 (0.50 g, 0.86 mmol) in dryTHF (10 mL) was added dropwise and the mixture was reacted at −78° C.for 15′. Ethylformate (1.27 g, 17.18 mmol) was then added and reactedfor 1 hr prior the reaction was allowed to warm to room temperature. Theraction mixture was poured into H₂O (50 mL) and extracted with EtOAc(3×50 mL). The combined organic layers were washed with brine (100 mL),dried over Na₂SO₄ anhydrous and evaporated under reduced pressure. Theintermediate 3 thus obtained was dissolved in MeOH (20 mL) and treatedat 0° C. with NaBH₄ for 30′. The reaction was quenched with H₂O (50 mL)and extracted with CH₂Cl₂ (3×50 mL). The combined organic layers werewashed with H₂O (100 mL), brine (100 mL), dried over Na₂SO₄ andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography (eluting with isopropanol in CHCl₃ from 2 to 7%,v/v) obtaining 0.29 g of compound 4 as epimeric mixture (0.48 mmol,56%).

3α,7α,12α-Trihydroxy-6α-ethyl-23(A)-fluoromethyl-fluoromethyl-5β-cholan-24-oicacid (Compound 6)

To a solution of compound 4 (0.09 g, 0.16 mmol) in dry CH₂Cl₂ (3 mL) asolution of DAST (0.04 g, 0.23 mmol) in dry CH₂Cl₂ (2 mL) was added. Themixture was reacted 2 hrs at −78° C. and subsequently poured into asaturated solution of NaHCO₃ (10 mL) and extracted with CH₂Cl₂ (2×15mL). The combined organic layers were washed with H₂O (10 mL), brine (10mL), dried over Na₂SO₄ anhydrous and evaporated under reduced pressure.The intermediate 5 was then dissolved in MeOH (10 mL) and treated withHCl 37% (0.3 mL) at room temperature for 12 hrs. The solvent wasevaporated under reduced pressure. The residue was suspended in H₂O (10mL) and extracted with CH₂Cl₂ (2×10 mL). The combined organic layerswere washed with H₂O (10 mL), brine (10 mL), dried over Na₂SO₄ anhydrousand evaporated under reduced pressure. The crude was dissolved in 2 mLof NaOH (3% in THF) and reacted at room temperature for 4 hrs. Thesolvent was evaporated under reduced pressure, suspended in H₂O (10 mL)and extracted with CH₂Cl₂ (2×10 mL). The combined organic layers werewashed with H₂O (10 mL), brine (10 mL), dried over Na₂SO₄ anhydrous andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography eluting with a solution of MeOH/CHCl₃ (98:2→9:1,v/v+0.1% AcOH) obtaining 12 mg of pure Compound 6 (0.026 mmol, 16%).

rf: 0.29 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeOH 20:80). ¹H-NMR(CD₃OD, 400 MHz) δ 0.73 (3H, s, 18-CH₃), 0.91-0.97 (6H, m,19-CH₃+CH₂CH₃), 1.05 (1H, d, J=6.0, 21-CH₃), 2.10-2.19 (2H, m, 22-CH₂),2.51-2.54 (1H, m, 23-CH), 3.41-3.45 (1H m, 3-CH), 3.53-3.57 (2H, m,CH₂F), 3.66 (1H, s, 7-CH), 3.97 (1H, s, 7-CH). ¹³C-NMR (CD₃OD, 100.6MHz) δ 12.0, 13.0, 18.1, 23.4, 24.0, 28.2, 29.1, 29.7, 30.7 (×2), 31.0,34.4, 36.0, 36.3, 36.7, 37.3, 41.7, 43.1 (×2), 46.9, 47.6, 58.9, 71.1,73.2, 74.0, 78.4 (J_(C-F)=392.3 Hz), 181.5.

Example 7: Synthesis of Compound 7

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(A+B)-hydroxymethyl-5β-cholan-24-oate(4)

To a solution of diisopropylamine (0.87 g, 8.59 mmol) in dry THF (25 mL)at −78° C., nBuLi 2.5 M in hexane (3.1 mL, 7.73 mmol) was addeddropwise. After 15′, a solution of compound 2 (0.50 g, 0.86 mmol) in dryTHF (10 mL) was added dropwise and the mixture was reacted at −78° C.for 15′. Ethylformate (1.27 g, 17.18 mmol) was then added and reactedfor 1 hr prior the reaction was allowed to warm to room temperature. Theraction mixture was poured into H₂O (50 mL) and extracted with EtOAc(3×50 mL). The combined organic layers were washed with brine (100 mL),dried over Na₂SO₄ anhydrous and evaporated under reduced pressure. Theintermediate 3 thus obtained was dissolved in MeOH (20 mL) and treatedat 0° C. with NaBH₄ for 30′. The reaction was quenched with H₂O (50 mL)and extracted with CH₂Cl₂ (3×50 mL). The combined organic layers werewashed with H₂O (100 mL), brine (100 mL), dried over Na₂SO₄ andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography (eluting with isopropanol in CHCl₃ from 2 to 7%,v/v) obtaining 0.29 g of compound 4 as epimeric mixture (0.48 mmol,56%).

3α,7α,12α-Trihydroxy-6α-ethyl-23(A)-fluoromethyl-fluoromethyl-5β-cholan-24-oicacid (Compound 7)

To a solution of compound 4 (0.09 g, 0.16 mmol) in dry CH₂Cl₂ (3 mL) asolution of DAST (0.04 g, 0.23 mmol) in dry CH₂Cl₂ (2 mL) was added. Themixture was reacted 2 hrs at −78° C. and subsequently poured into asaturated solution of NaHCO₃ (10 mL) and extracted with CH₂Cl₂ (2×15mL). The combined organic layers were washed with H₂O (10 mL), brine (10mL), dried over Na₂SO₄ anhydrous and evaporated under reduced pressure.The intermediate 5 was then dissolved in MeOH (10 mL) and treated withHCl 37% (0.3 mL) at room temperature for 12 hrs. The solvent wasevaporated under reduced pressure. The residue was suspended in H₂O (10mL) and extracted with CH₂Cl₂ (2×10 mL). The combined organic layerswere washed with H₂O (10 mL), brine (10 mL), dried over Na₂SO₄ anhydrousand evaporated under reduced pressure. The crude was dissolved in 2 mLof NaOH (3% in THF) and reacted at room temperature for 4 hrs. Thesolvent was evaporated under reduced pressure, suspended in H₂O (10 mL)and extracted with CH₂Cl₂ (2×10 mL). The combined organic layers werewashed with H₂O (10 mL), brine (10 mL), dried over Na₂SO₄ anhydrous andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography eluting with a solution of MeOH/CHCl₃ (98:2→9:1,v/v+0.1% AcOH) obtaining 16 mg of pure Compound 7 (0.034 mmol, 21%).

rf: 0.27 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeOH 20:80). ¹H-NMR(CD₃OD, 400 MHz) δ 0.72 (3H, s, 18-CH₃), 0.90-0.96 (6H, m,19-CH₃+CH₂CH₃), 1.06 (1H, d, J=6.3 Hz, 21-CH₃), 2.17-2.22 (1H, m,22-CH₂), 2.54-2.58 (1H, m, 23-CH), 3.28-3.33 (1H m, 3-CH), 3.38-3.58(2H, m, CH₂F), 3.66 (1H, s, 7-CH), 3.97 (1H, s, 7-CH). ¹³C-NMR (CD₃OD,100.6 MHz) δ 12.0, 13.0, 18.1, 23.5, 24.2, 28.2, 29.1, 29.7, 30.8, 31.0,34.4, 36.0, 36.3, 36.7, 37.3, 41.7, 43.1, 46.9, 47.4, 47.6, 58.9, 71.1,73.2, 74.0, 75.3 (J_(C-F)=452.7 Hz), 181.5.

Example 8: Synthesis of Compound 8

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

(E+Z)-3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-24,24-trimetylsilyloxy-methoxy-5β-chol-23-ene(3)

To a stirred solution of diisopropylamine (11.7 mL, 82.5 mmol) indistilled THF (40 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (32.0 mL, 79.3 mmol) was added dropwise. After 15′, thesolution was cooled up to −78° C. and chlorotrimethylsilane (12.7 mL,84.5 mmol) was added dropwise. After additional 15′, a solution of 3(6.0 g, 10.30 mmol) in distilled THF (20 mL) was added portionwise inabout 20′ maintaining the internal temperature not over −70° C. Once theaddition was completed, the reaction mixture was stirred at −78° C. for1 hr and then warmed at room temperature. Volatiles were removed underreduced pressure, and the residue was suspended in petroleum ether (80mL) and filtered under vacuum. The liquor was concentrated under reducedpressure, to give 10.12 g of oil residue that was used for the next stepwithout further purification.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(R)-hydroxy-5β-cholan-24-oate(4)

To a suspension of freshly crystallized and acetic acid freelead(IV)tetraacetate (6.85 g, 15.46 mmol) in distilled CH₂Cl₂ (50 mL)under N₂ atmosphere, a solution of 3 (10.12 g) in CH₂Cl₂ (30 mL) wasadded dropwise. After 30′ the reaction mixture was filtered under vacuumthrough a celite pad. The filtrate was concentrated under reducedpressure and the residue was filtered through a silica gel pad (h: 6 cm,φ: 2 cm) collecting the crude reaction mixture with petroleumether/AcOEt (8:2, v/v). After solvent evaporation, the residue (6.50 g)was dissolved in MeOH (50 mL) and treated with potassium carbonate (2.13g, 15.5 mmol) at room temperature for 15′. The mixture was then dilutedwith CH₂Cl₂ (50 mL) and filtered under vacuum. The filtrate was furtherdiluted with CH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueousphase was extracted with CH₂Cl₂ (3×40 mL), and the collected organiclayers were dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was purified by medium pressure liquidchromatography and collecting the desired compound with an isocraticelution constituted by petroleum ether/AcOEt (65:35, v/v) to obtain 4 in20% yield.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(S)-fluoro-5β-cholan-24-oate(5)

To a solution of 4 (0.92 g, 1.53 mmol) in distilled CH₂Cl₂ (40 mL) underN₂ atmosphere, diethylaminosulfurtrifluoride (1.0 mL, 7.7 mmol) wasadded and the reaction was stirred at room temperature for 10′. Themixture was cautiously poured in a saturated solution of NaHCO₃ (30 mL)and placed in a water-ice bath under magnetic stirring. Once the CO₂release was completed, the two phases were separated and the organiclayer was washed with H₂O (20 mL), brine (20 mL), dried over Na₂SO₄anhydrous and concentrated under reduced pressure. The residue waspurified by silica gel flash chromatography by using petroleumether/AcOEt (85:15, v/v) to give the desired compound 5 in nearlyquantitative yield.

¹H-NMR (CDCl₃, 400 MHz) δ 0.70 (3H, s, 18-CH₃), 0.87-0.92 (6H, m,19-CH₃+CH₂CH₃), 1.07 (3H, d, J=5.7 Hz, 21-CH₃), 3.30-3.37 (1H, m, 3-CH),3.35 (3H, s, OCH₂OCH₃), 3.36 (3H, s, OCH₂OCH₃), 3.43 (3H, s, OCH₂OCH₃),3.49 (1H, s, 7-CH), 3.79 (3H, s, CO₂CH₃), 3.81 (1H, s, 12-CH), 4.59-4.74(6H, m, 3×OCH₂OCH₃), 5.01 (1H, dd, J₁=10.1 Hz, J₂=52.0 Hz, 23-CHF).

3α,7α,12α-Trihydroxy-6α-ethyl-23(S)-fluoro-5β-cholan-24-oate (Compound8)

To a solution of 5 (0.92 g, 1.53 mmol) in MeOH (20 mL), HCl 3 N (4.6 ml,13.8 mmol) was added, and the mixture was stirred at 45° C. for 18 hrs.Sodium hydroxide (0.90 g, 22.95 mmol) was added, and the mixture wasstirred at 45° C. for additional 5 hrs. MeOH was removed under reducedpressure and the residue was diluted with H₂O up to 30 mL and washedwith Et₂O (2×15 mL). The aqueous phase was acidified with HCl 3 N,extracted with CH₃Cl₃/MeOH (85:15, v/v) (5×30 mL) and concentrated underreduced pressure. The residue was purified by RP-18 medium pressureliquid chromatography by using H₂O/MeOH as eluent (6:4→3:7) to obtainthe desired compound Compound 8 in 87% yield.

rf: 0.44 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(CD₃OD, 400 MHz) δ: 0.74 (3H, s, 18-CH₃), 0.89-0.92 (6H, m,19-CH₃+CH₂CH₃), 1.13 (3H, d, J=6.3 Hz, 21-CH₃), 2.17-2.23 (1H, m,22-CH₂), 3.34-3.34 (1H, m, 3-CH), 3.67 (1H, s, 7-CH), 3.97 (1H, s,12-CH), 4.99 (1H, psd, J_((H-F))=48 Hz, 23-CHF). ¹³C-NMR (CD₃OD, 100.6MHz) δ 12.9, 13.8, 19.7, 24.3 (×2), 25.0, 29.1, 29.7, 31.9, 35.3, 36.2,37.2 (×2), 37.6, 41.2 (d, J_(C-F)=19.8 Hz) 42.6, 44.0 (×2), 47.8, 48.5,72.0, 74.1, 74.9, 91.7 (d, J_(C-F) ⁼180.4 Hz), 176.7 (d, J_(C-F)=22.0Hz). ¹⁹F-NMR (DMSO-d6, 376.5 MHz) δ −184.7 (1F, m).

Example 9: Synthesis of Compound 9

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

(E+Z)-3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-24,24-trimetylsilyloxy-methoxy-5β-chol-23-ene(3)

To a stirred solution of diisopropylamine (11.7 mL, 82.5 mmol) indistilled THF (40 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (32.0 mL, 79.3 mmol) was added dropwise. After 15′, thesolution was cooled up to −78° C. and chlorotrimethylsilane (12.7 mL,84.5 mmol) was added dropwise. After additional 15′, a solution of 3(6.0 g, 10.30 mmol) in distilled THF (20 mL) was added portionwise inabout 20′ maintaining the internal temperature not over −70° C. Once theaddition was completed, the reaction mixture was stirred at −78° C. for1 hr and then warmed at room temperature. Volatiles were removed underreduced pressure, and the residue was suspended in petroleum ether (80mL) and filtered under vacuum. The liquor was concentrated under reducedpressure, to give 10.12 g of oil residue that was used for the next stepwithout further purification.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(S)-hydroxy-5β-cholan-24-oate(4)

To a suspension of freshly crystallized and acetic acid freelead(IV)tetraacetate (6.85 g, 15.46 mmol) in distilled CH₂Cl₂ (50 mL)under N₂ atmosphere, a solution of 3 (10.12 g) in CH₂Cl₂ (30 mL) wasadded dropwise. After 30′ the reaction mixture was filtered under vacuumthrough a celite pad. The filtrate was concentrated under reducedpressure and the residue was filtered through a silica gel pad (h: 6 cm,φ: 2 cm) collecting the crude reaction mixture with petroleumether/AcOEt (8:2, v/v). After solvent evaporation, the residue (6.50 g)was dissolved in MeOH (50 mL) and treated with potassium carbonate (2.13g, 15.5 mmol) at room temperature for 15′. The mixture was then dilutedwith CH₂Cl₂ (50 mL) and filtered under vacuum. The filtrate was furtherdiluted with CH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueousphase was extracted with CH₂Cl₂ (3×40 mL), and the collected organiclayers were dried over Na₂SO₄ anhydrous and concentrated under reducedpressure. The residue was purified by medium pressure liquidchromatography and collecting the desired compound with an isocraticelution constituted by petroleum ether/AcOEt (65:35, v/v) in 19% yield.

¹H-NMR (CDCl₃, 200 MHz) δ 0.65 (3H, s, 18-CH₃), 0.81-0.88 (6H, m,19-CH₃+CH₂CH₃), 0.99 (3H, d, J=6.4 Hz, 21-CH₃), 3.32-3.36 (1H, m, 3-CH),3.33 (6H, m, 2×OCH₂OCH₃), 3.39 (3H, s, OCH₂OCH₃), 3.46 (1H, s, 7-CH),3.74 (3H, s, CO₂CH₃), 3.76 (1H, s, 12-CH), 4.18 (1H, t, J=6.6 Hz,23-CH), 4.51-4.72 (6H, m, 3×OCH₂OCH₃). ¹³C-NMR (CDCl₃, 50.3 MHz) δ 11.7,12.4, 18.7, 22.8, 23.0, 23.8, 24.9, 27.3, 27.6, 27.9, 30.3, 33.5, 35.5(×2), 40.7, 41.2, 41.8, 42.2, 45.8, 46.3, 46.7, 52.2, 54.9, 55.7, 55.9,69.9, 77.4, 79.9, 80.0, 94.3, 95.8, 98.4, 176.0.

Methyl3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23(R)-fluoro-5β-cholan-24-oate(5)

To a solution of 4 (0.92 g, 1.53 mmol) in distilled CH₂Cl₂ (40 mL) underN₂ atmosphere, diethylaminosulfurtrifluoride (1.0 mL, 7.7 mmol) wasadded and the reaction was stirred at room temperature for 10′. Themixture was cautiously poured in a saturated solution of NaHCO₃ (30 mL)and placed in a water-ice bath under magnetic stirring. Once the CO₂release was completed, the two phases were separated and the organiclayer was washed with H₂O (20 mL), brine (20 mL), dried over Na₂SO₄anhydrous and concentrated under reduced pressure. The residue waspurified by silica gel flash chromatography by using petroleumether/AcOEt (85:15, v/v) to give the desired compound 5 in nearlyquantitative yield.

¹H-NMR (CDCl₃, 400 MHz) δ 0.69 (3H, s, 18-CH₃), 0.87-0.91 (6H, m,19-CH₃+CH₂CH₃), 1.05 (3H, d, J=5.7 Hz, 21-CH₃), 3.30-3.37 (1H, m, 3-CH),3.35 (3H, s, OCH₂OCH₃), 3.37 (3H, s, OCH₂OCH₃), 3.43 (3H, s, OCH₂OCH₃),3.49 (1H, s, 7-CH), 3.76 (1H, s, 12-CH), 3.79 (3H, s, CO₂CH₃), 4.59-4.75(6H, m, 3×OCH₂OCH₃), 4.97 (1H, dt, J₁=4.9 Hz, J₂=48.0 Hz, 23-CHF).

3α,7α,12α-Trihydroxy-6α-ethyl-23(R)-fluoro-5β-cholan-24-oate (Compound9)

To a solution of 5 (0.92 g, 1.53 mmol) in MeOH (20 mL), HCl 3 N (4.6 ml,13.8 mmol) was added, and the mixture was stirred at 45° C. for 18 hrs.Sodium hydroxide (0.90 g, 22.95 mmol) was added, and the mixture wasstirred at 45° C. for additional 5 hrs. MeOH was removed under reducedpressure and the residue was diluted with H₂O up to 30 mL and washedwith Et₂O (2×15 mL). The aqueous phase was acidified with HCl 3 N,extracted with CH₃Cl₃/MeOH (85:15, v/v) (5×30 mL) and concentrated underreduced pressure. The residue was purified by RP-18 medium pressureliquid chromatography by using H₂O/MeOH as eluent (6:4→3:7) to obtainthe desired compound Compound 9 in 82% yield.

rf: 0.42 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(CD₃OD, 400 MHz) δ: 0.74 (3H, s, 18-CH₃), 0.89-0.91 (6H, m,19-CH₃+CH₂CH₃), 1.11 (3H, d, J=5.0 Hz, 21-CH₃), 2.17-2.21 (1H, m,22-CH₂), 3.31-3.35 (1H, m, 3-CH), 3.66 (1H, s, 7-CH), 3.98 (1H, s,12-CH), 5.01 (1H, dd, J_(1(H-F))=10.0 Hz, J_(2(H-F))=51.3 Hz, 23-CHF).¹³C-NMR (CD₃OD, 100.6 MHz) δ: 12.94, 13.87, 18.40, 24.40 (×2), 25.04,29.17, 29.69, 30.63, 31.95, 34.63, 35.29, 37.20 (×2), 37.58, 40.90 (d,J_(C-F)=20.5 Hz), 42.62, 44.02, 44.10, 47.85, 48.55, 72.00, 74.07,74.86, 89.01 (d, J_(C-F)=180.4 Hz), 175.43 (d, J_(C-F)=24.1 Hz). ¹⁹F-NMR(DMSO-d6, 376.5 MHz) δ −184.0 (1F, bs).

Example 10: Synthesis of Compound 10

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-5-cholan-24-oate (2)

To a solution of OCA (1) (1.93 g, 4.59 mmol) in MeOH (30 mL),p-toluensulfonic acid (0.09 g, 0.46 mmol) was added, and the resultingmixture was reacted under ultrasounds irradiation for 2 hrs. MeOH wasremoved under reduced pressure, and the residue was dissolved in AcOEt(30 mL) and washed with a saturated solution of NaHCO₃ (30 mL), H₂O (30mL) and brine (20 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was then dissolvedin CH₂Cl₂ (60 mL), and treated with diisopropylethylamine (7.1 mL, 41.4mmol), 4-(N,N-dimethylamino)-pyridine (0.05 g, 0.46 mmol) andmethoxymethylchloride (2.1 mL, 27.6 mmol). The mixture was then refluxedfor 36 hrs. The reaction was cooled at room temperature and washed withH₂O (30 mL), HCl 3 N (30 mL), H₂O (30 mL), saturated NaHCO₃ (300 mL) andbrine (30 mL). The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure, to afford 2.38 g (4.55 mmol) of 2as pale yellow oil (quantitative yield).

Methyl3α,7α-dimethoxymethyloxy-6α-ethyl-23(R+S)-hydroxy-5β-cholan-24-oate (4)

To a stirred solution of diisopropylamine (7.6 mL, 82.5 mmol) indistilled THF (30 mL) under N₂ atmosphere and cooled at −40° C., nBuLi2.5 M in hexane (20.6 mL, 51.6 mmol) was added dropwise. After 15′, thesolution was cooled to −78° C. and chlorotrimethylsilane (8.5 mL, 67.0mmol) was added dropwise. After additional 15′, a solution of 2 (3.50 g,6.70 mmol) in distilled THF (20 mL) was added portionwise in about 20′,maintaining the internal temperature at −70° C. Once the addition wasfinished, the reaction mixture was stirred at −78° C. for 1 hr and thenwarmed at room temperature. Volatiles were removed under reducedpressure. The residue was suspended in petroleum ether (80 mL) andfiltered under vacuum. The liquor was concentrated under reducedpressure and dissolved in distilled CH₂Cl₂ (30 mL). The resultingsolution was added dropwise to a suspension of freshly crystallized andacetic acid free lead(IV)tetraacetate (4.45 g, 10.50 mmol) in distilledCH₂Cl₂ (50 mL) under N₂ atmosphere. After 30′ the reaction mixture wasfiltered under vacuum through a celite pad. The filtrate wasconcentrated under reduced pressure, and the residue was filteredthrough a silica gel pad (h: 4 cm, φ: 2 cm), collecting the crude withpetroleum ether/AcOEt (8:2, v/v). After solvent evaporation, the residuewas dissolved in MeOH (30 mL) and treated with potassium carbonate (1.38g, 10.05 mmol). The resulting suspension was vigorously stirred at roomtemperature for 15′. The mixture was then diluted with CH₂Cl₂ (40 mL)and filtered under vacuum. The filtrate was diluted with additionalCH₂Cl₂ (70 mL) and washed with brine (70 mL). The aqueous phase wasextracted with CH₂Cl₂ (3×40 mL), and all the collected organic layerswere dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography byusing petroleum ether/AcOEt (8:2→1:1, v/v) to afford 1.73 g (3.21 mmol,48%) of 4 (as epimeric mixture).

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-23-oxo-5β-cholan-24-oate (5)

To a solution of oxalyl chloride (40.2 mL, 2.10 mmol) in distilledCH₂Cl₂ (12 mL) under N₂ atmosphere and cooled ad −60° C., DMSO (0.30 mL,4.18 mmol) diluted in CH₂Cl₂ (2 mL) was added dropwise. After 15′ asolution of 4 (0.45 g, 0.84 mmol) in CH₂Cl₂ (12 mL) was added dropwise,and the resulting mixture was stirred at −60° C. for 1 hr. Triethylamine(1.2 mL, 8.40 mmol) was added dropwise, and the mixture was slowlywarmed at room temperature. The reaction mixture was treated with KOH 1M (20 mL) for 5′, the two phases were separated and the aqueous one wasextracted with CH₂Cl₂ (2×20 mL). The collected organic layers were driedover anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by silica gel flash flash chromatography usingpetroleum ether/AcOEt (9:1→8:2, v/v) as eluent to give 5 (0.43 g, 0.80mmol, 96%).

Methyl3α,7α-dimethoxymethyloxy-6α-ethyl-23,23-gemdifluoro-5β-cholan-24-oate(6)

To a solution of 5 (0.43 g, 0.80 mmol) in distilled CH₂Cl₂ (20 mL) underN₂ atmosphere, diethylaminosulfurtrifluoride (1.06 mL, 8.02 mmol) wasadded, and the reaction was stirred at room temperature for 12 hrs. Themixture was cautiously poured in saturated NaHCO₃ (50 mL) and stirred ina water-ice bath until CO₂ release completation. The two phases wereseparated and the organic layer was washed with H₂O (20 mL), brine (20mL), dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography byusing a solution of petroleum ether/AcOEt (95:5→8:2, v/v) to yield 0.31g (0.56 mmol, 71%) of pure 6.

3α,7α-Dihydroxy-6α-ethyl-23,23-gemdifluoro-5β-cholan-24-oic acid(Compound 10)

To a solution of 6 (0.31 g, 0.56 mmol) in MeOH (15 mL), HCl 3 N (1.7 mL,5.04 mmol) was added, and the mixture was stirred at 45° C. for 12 hrs.Sodium hydroxide (0.33 g, 8.40 mmol) was added, and the mixture wasstirred at 45° C. for additional 4 hrs. MeOH was removed under reducedpressure, and the residue was diluted with H₂O up to 15 mL and washedwith Et₂O (2×10 mL). The aqueous phase was acidified by adding HCl 3 N,and the resulting whitish suspension was filtered through a RP-18 silicagel pad (h: 3 cm, φ: 1 cm) under vacuum, washing with H₂O (50 mL) andcollecting the crude material using a solution of H₂O/MeCN 40:60 (v/v).Once the solvent was removed under reduced pressure, the residue waspurified by RP-18 medium pressure liquid chromatography with H₂O/MeCN(8:2→4:6, v/v). 0.22 g (0.48 mmol, 86%) of the pure difluoro derivativeCompound 10 was obtained.

rf: 0.31 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 60:40). ¹H-NMR(DMSO-d₆, 400 MHz) δ: 0.62 (3H, s, 18-CH₃), 0.82-0.91 (6H, m,19-CH₃+CH₂CH₃), 1.01 (3H, d, J=6.1 Hz, 21-CH₃), 2.09-2.13 (1H, m,22-CH₂), 3.17-3.21 (1H, m, 3-CH), 3.49 (1H, s, 7-CH), 4.07 (1H, bs, OH).¹³C-NMR (CD₃OD, 100.6 MHz) δ 11.4, 11.6, 19.6, 20.3, 22.1, 22.9, 23.0,28.0, 30.4, 30.9, 32.6, 33.5, 35.1, 35.5, 41.2, 42.0, 45.3, 48.5, 50.2,55.6, 68.3, 70.5, 117.3 (J_(C-F)=248.7 Hz), 165.6 (J_(C-F)=31.6 Hz).¹⁹F-NMR (DMSO-d6, 376.5 MHz) δ −100.9 (2F, m). MS-TIC (−) m/z: 455.4.

Example 11: Synthesis of Compound 11

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₁₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-amide (3)

2 (1.55 g, 3.44 mmol) was treated with NaOH 5% in MeOH (30 mL) at refluxunder magnetic stirring for 2 hrs. MeOH was removed and the residue wasdissolved in AcOEt (50 mL) and washed with H₂O (50 mL) and brine (50mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The oil residue was dissolved in THF (30 mL) andtreated with ethyl chloroformiate (0.45 mL, 4.82 mmol) and triethylamine(0.72 mL, 5.16 mmol). The mixture was vigorously stirred for 1 hr. Thereaction was diluted with AcOEt (50 mL), washed with H₂O (30 mL),aqueous HCl 1 N (30 mL), brine (30 mL), dried over Na₂SO₄ anhydrous andconcentrated under reduced pressure, to obtain the desired amideintermediate 3 in quantitative yield. The crude was used for the nextstep without further purification.

3α,7α,12α-trimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (4)

To a solution of 3 (1.95 g, 3.44 mmol) in DMF (20 mL), cyanuric chloride(0.42 g, 6.88 mmol) was added and the reaction was stirred at roomtemperature for 18 hrs. The mixture was poured into AcOEt (100 mL) andwashed with H₂O (5×50 mL), brine (30 mL), dried over Na₂SO₄ anhydrousand concentrated under reduced pressure. The residue was purified bysilica gel flash chromatography with petroleum ether/AcOEt (9:1→7:3,v/v) to get 1.15 g (2.10, mmol, 61%) of the cyano derivative 4.

3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-24-N-hydroxy-cholanamidine (5)

To a solution of 4 (0.60 g, 1.11 mmol) in EtOH (30 mL), hydroxylaminechlorohydrate (0.77 g, 11.16 mmol) and sodium carbonate decahydrate(3.20 g, 11.16 mmol) were added and the mixture was refluxed for 36 hrs.Volatiles were removed under reduced pressure and the resulting residuewas dissolved in EtOAc (30 mL), washed with H₂O (3×30 mL), brine (30mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure. The crude was purified by silica gel flashchromatography by using CHCl₃/MeOH (98:2→95:5, v/v) thereby obtaining0.42 g (0.72 mmol, 65%) of pure 5.

3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-24-N[(ethoxycarbonyl)oxy]imidocholanamide (6)

To a solution of 5 (0.42 g, 0.72 mmol) in distilled CH₂Cl₂ (30 mL),cooled at 0° C. and under N₂ atmosphere, ethyl chloroformate (0.07 mL,0.94 mol) and pyridine (0.09 mL, 1.08 mmol) were added dropwise and thereaction mixture was stirred at room temperature for 1 hr. The reactionwas quenched with H₂O (15 mL), the two phases were separated and theorganic layer was washed with H₂O (3×15 mL), brine (15 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure toafford 6 as crude material (0.44 g), which was used for the next stepwithout further purification.

3α,7α,12α-trimethoxymethyloxy-6α-ethyl-24-5β-23([1,2,4]-oxadiazole-3-one-5yl)-cholane(7)

A solution of 6 (0.44 g) in toluene (20 mL) and pyridine (5 mL) wasrefluxed for 48 hrs. The mixture was then diluted with AcOEt (50 mL),washed with H₂O (3×50 mL), brine (30 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure, to obtain 0.43 g of 7which was used as such for the next step.

3α,7α,12α-trihydroxy-6α-ethyl-24-nor-5β-23([1,2,4]-oxadiazole-3-one-5yl)-cholane(Compound 11)

To a solution of crude 8 (0.43 g) in acetone (15 mL), HCl 3 N (5 mL) wasadded, and the mixture was stirred at 50° C. for 6 hrs. The organicsolvent was removed under reduced pressure, the residue was dissolved inCHCl₃ (30 mL) and washed with H₂O (3×20 mL), brine (20 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel flash chromatography, by usingCHCl₃/MeOH/AcOH (98:2:0.1-93:7:0.1, v/v/v), to give 0.14 g (0.29 mmol,41% from intermediate 6) of pure Compound 11.

rf: 0.37 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(CDCl₃, 400 MHz) δ: 0.70 (3H, s, 18-CH₃), 0.89-0.92 (6H, m,19-CH₃+CH₂CH₃), 1.03 (3H, d, J=5.4 Hz, 21-CH₃), 2.18-2.59 (2H, m,23-CH₂), 3.42-3.45 (1H, m, 3-CH), 3.71 (1H, s, 7-CH), 3.99 (1H, s,12-CH). ¹³C-NMR (CDCl₃, 100.6 MHz) δ: 11.6, 12.4, 17.2, 21.8, 22.1,22.7, 23.2, 26.7, 27.5, 28.2, 30.0, 31.8, 33.4, 35.1, 35.4 (×2), 39.9,41.3, 41.8, 45.0, 46.2, 46.4, 71.0, 72.2, 73.3, 160.4, 160.9.

Example 12: Synthesis of Compound 12

Methyl 3α,7α,12α-trimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of 3α,7α,12α-trihydroxy-6α-ethyl-5β-cholan-24-oic acid(6-ECA, 1) (20.0 g, 45.9 mmol) in MeOH (150 mL), p-toluensulfonic acid(0.44 g, 2.29 mmol) was added and the resulting mixture was reactedunder ultrasound irradiation for 2 hrs. MeOH was removed under reducedpressure and the residue was dissolved in AcOEt (200 mL) and washed witha saturated solution of NaHCO₃ (200 mL), H₂O (200 mL) and brine (200mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was then dissolved in CH₂Cl₂ (180mL) and the resulting solution was treated with diisopropylethylamine(94 mL, 550.5 mmol), 4-(N,N-dimethylamino)-pyridine (0.56 g, 4.6 mmol)and methoxymethylchloride (31.2 mL, 412.8 mmol). The mixture was stirredand refluxed for 48 hrs. The reaction was cooled at room temperature andwashed with H₂O (100 mL), HCl 3 N (100 mL), H₂O (100 mL), saturatedNaHCO₃ (100 mL) and brine (100 mL). The organic layer was dried overNa₂SO₄ anhydrous and concentrated under reduced pressure to afford 26.61g (45.65 mmol) of 2 as a pale yellow oil (quantitative yield).

3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-5β-cholan-24-amide (3)

2 (1.55 g, 3.44 mmol) was treated with NaOH 5% in MeOH (30 mL) at refluxunder magnetic stirring for 2 hrs. MeOH was removed and the residue wasdissolved in AcOEt (50 mL) and washed with H₂O (50 mL) and brine (50mL). The organic layer was dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The oil residue was dissolved in THF (30 mL) andtreated with ethyl chloroformiate (0.45 mL, 4.82 mmol) and triethylamine(0.72 mL, 5.16 mmol). The mixture was vigorously stirred for 1 hr. Thereaction was diluted with AcOEt (50 mL), washed with H₂O (30 mL),aqueous HCl 1 N (30 mL), brine (30 mL), dried over Na₂SO₄ anhydrous andconcentrated under reduced pressure, to obtain the desired amideintermediate 3 in quantitative yield. The crude was used for the nextstep without further purification.

3α,7α,12α-Trimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (4)

To a solution of 3 (1.95 g, 3.44 mmol) in DMF (20 mL), cyanuric chloride(0.42 g, 6.88 mmol) was added and the reaction was stirred at roomtemperature for 18 hrs. The mixture was poured into AcOEt (100 mL) andwashed with H₂O (5×50 mL), brine (30 mL), dried over Na₂SO₄ anhydrousand concentrated under reduced pressure. The residue was purified bysilica gel flash chromatography with petroleum ether/AcOEt (9:1→7:3,v/v) to get 1.15 g (2.10, mmol, 61%) of the cyano derivative 4.

3α,7α,12α-Trihydroxy-6α-ethyl-23-(tetrazol-5-yl)-24-nor-5β-cholane(Compound 12)

To a solution of 4 (0.20 g, 0.36 mmol) in distilled PhMe (10 mL) andunder N₂ atmosphere, tributyltin azide (0.50 mL, 1.80 mmol) was addedand the resulting mixture was refluxed for 72 hrs. When completed, thereaction mixture was diluted with EtOAc (50 mL), washed with H₂O (3×15mL), brine (15 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude 5 (0.24 g) was dissolvedin acetone (15 mL) and treated with HCl 3 N (5 mL) at 50° C. for 6 hrs.Acetone was removed under reduced pressure, the residue was diluted withH₂O (20 mL) and basified up to pH 14 by adding NaOH 3 N. The mixture waswashed with Et₂O (3×20 mL), acidified with HCl 3 N, extracted withCHCl₃/MeOH (9:1, v/v), dried over Na₂SO₄ anhydrous and concentratedunder reduced pressure. The residue was purified by silica gel flashchromatography using CHCl₃/MeOH/AcOH (96:4:0.1→90:10:0.1, v/v/v), to get0.11 g (0.24 mmol, 66%) of pure Compound 12.

rf: 0.39 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(CD₃OD, 400 MHz) δ: 0.70 (3H, s, 18-CH₃), 0.88-0.91 (6H, m,19-CH₃+CH₂CH₃), 1.13 (3H, d, J=6.1 Hz, 21-CH₃), 2.18-2.22 (1H, m,22-CH₂), 2.83-2.91 (1H, m, 23-CH₂), 2.97-3.04 (1H, m, 23-CH₂), 3.29-3.34(1H, m, 3-CH), 3.66 (1H, s, 7-CH), 3.96 (1H, s, 12-CH). ¹³C-NMR (CD₃OD,100.6 MHz) δ: 12.0, 12.9, 17.5, 21.1, 23.4, 23.5, 24.1, 28.2, 28.7,29.7, 31.0, 34.3, 35.2, 36.3, 36.6, 36.7, 41.7, 43.1, 43.1, 46.9, 47.5,47.8, 71.1, 73.1, 74.0, 158.8.

Example 13: Synthesis of Compound 13

Methyl 3α,7α-dihydroxy-6α-ethyl-5β-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with saturated NaHCO₃ (100 mL), H₂O (100 mL), brine (100mL), dried over anhydrous Na₂SO₄ and evaporated under reduced pressure.The white solid thus obtained (5.17 g, 11.89 mmol) was used for the nextstep without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-5β-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL), the combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5β-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-Diacetoxy-6α-ethyl-24-nor-5β-cholan-23-amide (6)

To a solution of acid 5 (2.12 g, 4.31 mmol) in dry THF (40 mL) at 0° C.,triethylamine (0.65 g, 6.47 mmol) and ethylchloroformate (0.65 g, 6.04mmol) were added. The resulting suspension was stirred at roomtemperature for 1 hr. NH₃ (28% in H₂O, 0.73 g, 2.94 mL) was addeddropwise to the mixture and stirred at room temperature for 12 hrs. Themixture was poured into H₂O (50 mL) and extracted with EtOAc (2×50 mL).The combined organic layers were washed with HCl 1 N (50 mL), H₂O (50mL), brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The compound 6 was used for the next step withoutfurther purification.

3α,7α-Diacetoxy-6α-ethyl-22-cyano-23,24-bisnor-5β-cholane (7)

To a solution of amide 6 (1.50 g, 3.06 mmol) in DMF (30 mL), cyanogenchloride (0.37 g, 6.013 mmol) was added and the reaction mixture wasstirred at room temperature for 12 hrs. The mixture was diluted withEtOAc (30 mL), washed with H₂O (3×30 mL), brine (30 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The oilyresidue was purified by silica gel flash flash chromatography elutingwith EtOAc in petroleum ether from 10 to 50% (v/v) obtaining 0.98 g(2.08 mmol, 68%) of the desired nitrile derivative 7.

3α,7α-dimethoxymethyloxy-6α-ethyl-22-[1-(tributylstannyl)-tetrazol-5-yl]-23,24-bisnor-5β-cholane(8)

To a solution of nitrile 7 (0.81 g, 1.72 mmol) in toluene (25 mL),tributyltin azide (2.87 g, 8.58 mmol) was added ant the reaction wasrefluxed for 36 hrs. The mixture was then cooled at room temperature,diluted with EtOAc (25 mL), washed with HCl 3 N (3×20 mL), H₂O (50 mL),brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure dried over Na₂SO₄ and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography(eluting with methanol in dichloromethane from 2 to 5%, v/v) obtaining0.31 g (0.61 mmol, 61%) of the desired protected tetrazole 8.

3α,7α-Dihydroxy-6α-ethyl-23-(tetrazol-5-yl)-24-nor-5β-cholane (Compound13)

To a suspension of tetrazole 8 (0.27 g, 0.53 mmol) in H₂O (1 mL) andMeOH (7 mL), KOH (0.444 g, 7.87 mmol) was added. The mixture wassubmitted to microwave irradiation (T=135° C., P_(max)=250 psi,Power_(max)=200 W) for 16 hrs. The organic solvent was removed underreduced pressure, the residue was dissolved in H₂O (50 mL) and extractedwith Et₂O (2×15 mL). The aqueous phase was acidified with HCl 3 N andextracted with CH₂Cl₂ (3×15 mL). The combined organic layers were washedwith H₂O (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The crude was purified by silica gelflash chromatography (eluting with MeOH in CHCl₃ from 0 to 10%, v/v) tofurnish the desired derivative Compound 13 as white solid (0.19 g, 0.44mmol, 84%).

rf: 0.31 (TLC: Silica Gel 60 F₂₅₄S; eluent: CHCl₃/MeOH/AcOH 96:4:1).¹H-NMR (CD₃OD, 400 MHz) δ 0.75 (3H, s, 18-CH₃), 0.89-0.892 (9H, m,19-CH₃+21-CH₃+CH₂CH₃), 2.66 (1H, dd, J₁=9.7 Hz, J₂=14.5 Hz, 22-CH₂),3.04 (1H, dd, J₁=3.2 Hz, J₂=14.5 Hz, 22-CH₂), 3.29-3.35 (1H, m, 3-CH),3.67 (1H, s, 7-CH). ¹³C-NMR (CD₃OD, 400 MHz) δ 12.9, 13.1, 20.1, 22.8,24.4, 24.6, 25.5, 30.4, 31.9, 32.1, 35.2, 35.4, 37.5, 37.6, 38.4, 41.7,42.1, 44.0, 44.7, 47.8, 52.9, 58.3, 72.0, 74.0, 158.1.

Example 14: Synthesis of Compound 14

3α-Acetoxy-6α-ethyl-7α-hydroxy-5β-cholan-24-oic acid (2)

To a solution of OCA (1) (5.00 g, 11.87 mmol) in CH₂Cl₂ (50 mL), Ac₂O(8.4 mL, 89.22 mmol), diisopropylethylamine (15.5 mL, 89.22 mmol) and4-(N,N-dimethylamino)-pyridine (0.54 g, 4.46 mmol) were added, and theresulting suspension was stirred at reflux for 10′. The mixture wascooled at room temperature, diluted with CH₂Cl₂ (50 mL) and washed withH₂O (3×50 mL) and HCl 3 N (50 mL). The organic layer was treated withHCl 37% (5 mL) for 2′. H₂O (50 mL) was added, the two phases separatedand the organic one was washed with saturated NaHCO₃ (100 mL) and brine(100 mL). The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure, to afford 5.49 g of 2 as paleyellow solid (quantitative yield).

3α-Acetoxy-6α-ethyl-7α-oxo-5β-cholan-24-oic acid (3)

To a solution of 2 (5.49 g, 11.87 mmol) in CH₂Cl₂ (60 mL), pyridiniumchlorochromate (7.67 g, 35.69 mmol) was added, and the resulting darkmixture was stirred at room temperature for 2 hrs. The thus obtainedsuspension was filtered under vacuum through a celite pad, and thefiltrate was washed with saturated NaHCO₃ (100 mL), brine (100 mL),dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by silica gel flash chromatography, by usingpetroleum ether/AcOEt (8:2→6:4, v/v), thereby obtaining 4.43 g (9.61mmol, 80%) of pure 3.

3α-Acetoxy-6α-ethyl-7α-oxo-22-cyano-23,24-bisnor-5β-cholane (4)

To a solution of 3 (4.43 g, 9.55 mmol) in trifluoroacetic acid (30 mL)cooled at 0° C., trifluoroacetic anhydride (10.1 mL, 71.61 mmol) wasadded, and the resulting mixture was stirred at the same temperature for45′. Keeping the temperature at 0° C., sodium nitrite (1.98 g, 28.64mmol) was added portionwise, and the thus obtained red solution wasstirred at 0° C. for 1 hr and then at 45° C. for additional 50′. Themixture was cooled at room temperature and slowly poured in H₂O/ice bath(about 150 mL) and extracted with AcOEt (3×50 mL). The collected organiclayers were washed with NaOH 5 M (3×50 mL) till neutral pH, washed withH₂O (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure, to obtain 3.51 g of 4 that was used as such forthe next step.

3α-Acetoxy-6α-ethyl-7α-hydroxy-22-cyano-23,24-bisnor-5β-cholane (5)

To a solution of 4 (3.51 g, 8.18 mmol) in THF (80 mL) and H₂O (20 mL)cooled at 0° C., NaBH₄ (1.25 g, 32.88 mmol) was added portionwise. After30′ the reaction was quenched by adding AcOEt (100 mL) and HCl 3 N (30mL). The two phases were separated, and the aqueous one was extractedwith AcOEt (2×50 mL). The collected organic layers were washed with H₂O(50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure, to afford the desired 5 (3.52 g) in quantitativeyield.

3α,7α-Dihydroxy-6α-ethyl-24-nor-5β-23-N-hydroxy-cholanamidine (6)

To a solution of 5 (3.52 g, 8.18 mmol) in EtOH (120 mL), hydroxylaminechlorohydrate (17.55 g, 109.35 mmol) and sodium carbonate decahydrate(31.28 g, 109.35 mmol) were added and the mixture was stirred andrefluxed for 48 hrs. Volatiles were removed under reduced pressure andthe resulting residue was dissolved in EtOAc (150 mL), washed with H₂O(3×100 mL), brine (100 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure, to obtain 3.45 g of 6, which wasused for the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24-nor-5β-23-N[(ethoxycarbonyl)oxy]imidocholanamide(7)

To a solution of 6 (3.45 g, 8.18 mmol mmol) in distilled CH₂Cl₂ (100mL), cooled at 0° C. and under N₂ atmosphere, pyridine (0.99 mL, 12.27mmol) and ethyl chloroformate (0.70 mL, 7.36 mmol) were added dropwiseand the reaction mixture was stirred at room temperature for 1 hr. Thereaction was quenched with H₂O (50 mL), the two phases were separatedand the organic layer was washed with H₂O (3×50 mL), brine (50 mL),dried over anhydrous Na₂SO₄, filtered and concentrated under reducedpressure to afford 7 (3.50 g), which was used for the next step withoutfurther purification.

3α,7α-Dihydroxy-6α-ethyl-23,24-bisnor-5β-22([1,2,4]-oxadiazole-3-one-5yl)-cholane(Compound 14)

A solution of crude 7 (3.50 g) in toluene (100 mL) and pyridine (20 mL)was refluxed for 72 hrs. The mixture was then cooled at roomtemperature, diluted with AcOEt (200 mL), washed with H₂O (100 mL), HCl3 N (100 mL), H₂O (100 mL), brine (30 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure. The residue waspurified by silica gel flash chromatography by using a solution ofCH₂Cl₂/MeOH/AcOH (98:2:0.1→95:5:0.1, v/v/v), to obtain 1.23 g of pureCompound 14 in 34% from intermediate 5.

rf: 0.18 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeOH 20:80). ¹H-NMR(CD₃OD, 400 MHz) δ 0.75 (3H, s, 18-CH₃), 0.92-0.96 (6H, m,19-CH₃+CH₂CH₃), 0.99 (3H, d, J=5.8, 21-CH₃), 2.19 (1H, m, 22-CH₂), 2.67(1H, m, 22-CH₂), 3.28-3.35 (1H, m, 3-CH), 3.66 (1H, s, 7-CH). ¹³C-NMR(CD₃OD, 400 MHz) δ 12.0, 12.4, 19.1, 21.9, 23.4, 23.7, 24.5, 29.3, 31.2,32.8, 34.3, 34.5, 35.6, 36.6, 36.7, 40.8, 41.5, 43.1, 43.9, 46.9, 51.6,57.5, 71.1, 73.1, 160.7, 162.3.

Example 15: Synthesis of Compound 15

Methyl 3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of OCA (1) (1.93 g, 4.59 mmol) in MeOH (30 mL),p-toluensulfonic acid (0.09 g, 0.46 mmol) was added, and the resultingmixture was reacted under ultrasounds irradiation for 2 hrs. MeOH wasremoved under reduced pressure, and the residue was dissolved in AcOEt(30 mL) and washed with a saturated solution of NaHCO₃ (30 mL), H₂O (30mL) and brine (20 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was then dissolvedin CH₂Cl₂ (60 mL), and treated with diisopropylethylamine (7.1 mL, 41.4mmol), 4-(N,N-dimethylamino)-pyridine (0.05 g, 0.46 mmol) andmethoxymethylchloride (2.1 mL, 27.6 mmol). The mixture was then refluxedfor 36 hrs. The reaction was cooled at room temperature and washed withH₂O (30 mL), HCl 3 N (30 mL), H₂O (30 mL), saturated NaHCO₃ (300 mL) andbrine (30 mL). The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure, to afford 2.38 g (4.55 mmol) of 2as pale yellow oil (quantitative yield).

3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-cholan-24-amide (3)

2 (2.24 g, 4.31 mmol) was treated with 40 mL of a methanolic solution ofNaOH 5% at reflux under magnetic stirring for 2 hrs. MeOH was thenremoved, the residue was dissolved in AcOEt (60 mL) and washed with H₂O(60 mL) and brine (60 mL). The organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The oil residue wasdissolved in THF (40 mL) and treated with ethyl chloroformiate (0.57 mL,6.04 mmol) and triethylamine (0.90 mL, 6.47 mmol). The mixture wasvigorously stirred for 1 hr. Once the reaction was completed, themixture was diluted with AcOEt (60 mL), washed with H₂O (30 mL), HCl 1 N(30 mL), brine (30 mL) dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure, to obtain the desired intermediate 3 inquantitative yield. The crude was used for the next step without furtherpurification.

3α,7α-dimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (4)

To a solution of 3 (2.01 g, 3.96 mmol) in DMF (40 mL), cyanuric chloride(0.48 g, 7.92 mmol) were added, and the reaction was stirred at roomtemperature for 16 hrs. The mixture was poured into AcOEt (100 mL),washed with H₂O (5×50 mL) and brine, (30 mL) dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was purified bysilica gel flash chromatography with petroleum ether/AcOEt as eluent(9:1→65:35, v/v), to obtain 1.43 g (2.93 mmol, 74%) of 4.

3α,7α-Dihydroxy-6α-ethyl-23-(tetrazol-5-yl)-6α-ethyl-24-nor-5β-cholane(Compound 15)

To a solution of 4 (0.70 g, 1.43 mmol) in distilled PhMe (15 mL) andunder N₂ atmosphere, tributyltin azide (1.97 ml, 7.45 mmol) was addedand the resulting mixture was refluxed for 48 hrs. When completed, thereaction mixture was diluted with EtOAc (40 mL), washed with H₂O (3×20mL), brine (20 mL), dried over anhydrous Na₂SO₄, filtered andconcentrated under reduced pressure. The crude 5 (0.80 g) was dissolvedin acetone (30 mL) and HCl 3 N (10 mL), and the resulting mixture wasstirred at 50° C. for 6 hrs. Acetone was removed under reduced pressure,the residue was diluted with H₂O (20 mL) and basified up to pH 14 byadding aqueous NaOH 3 N. The mixture was washed with Et₂O (3×20 mL),acidified with HCl 3 N, extracted with a solution of CHCl₃/MeOH (95:15,v/v), dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The residue was purified by silica gel flash chromatography byusing an eluent constituted by CHCl₃/MeOH/AcOH (98:2:0.1→94:17:0.1,v/v/v), to give 0.36 g (0.81 mmol, 57% from intermediate 4) of pureCompound 15.

rf: 0.38 (TLC: Silica Gel F₂₅₄S; eluent: CH₂Cl₂/MeOH/AcOH 90:10:1).¹H-NMR (CD₃OD, 400 MHz) δ: 0.68 (3H, s, 18-CH₃), 0.89-0.93 (6H, m,19-CH₃+CH₂CH₃), 1.03 (3H, d, J=5.5 Hz, 21-CH₃), 2.87-2.92 (1H, m,23-CH₂), 2.97-3.00 (1H, m, 23-CH₂), 3.33-3.37 (1H, m, 3-CH), 3.65 (1H,s, 7-CH). ¹³C-NMR (CDCl₃, 100.6 MHz) δ: 12.9, 13.1, 19.6, 21.9, 22.9,24.4, 24.6, 25.4, 30.2, 32.1, 35.3, 35.4, 36.0, 37.5, 37.6, 41.9, 42.4,44.0, 44.7, 47.8, 52.5, 58.0, 72.0, 74.1, 159.2.

Example 16: Synthesis of Compound 16

Methyl 3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of OCA (1) (1.93 g, 4.59 mmol) in MeOH (30 mL),p-toluensulfonic acid (0.09 g, 0.46 mmol) was added, and the resultingmixture was reacted under ultrasounds irradiation for 2 hrs. MeOH wasremoved under reduced pressure, and the residue was dissolved in AcOEt(30 mL) and washed with a saturated solution of NaHCO₃ (30 mL), H₂O (30mL) and brine (20 mL). The organic layer was dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was then dissolvedin CH₂Cl₂ (60 mL), and treated with diisopropylethylamine (7.1 mL, 41.4mmol), 4-(N,N-dimethylamino)-pyridine (0.05 g, 0.46 mmol) andmethoxymethylchloride (2.1 mL, 27.6 mmol). The mixture was then refluxedfor 36 hrs. The reaction was cooled at room temperature and washed withH₂O (30 mL), HCl 3 N (30 mL), H₂O (30 mL), saturated NaHCO₃ (300 mL) andbrine (30 mL). The organic layer was dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure, to afford 2.38 g (4.55 mmol) of 2as pale yellow oil (quantitative yield).

3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-cholan-24-amide (3)

2 (2.24 g, 4.31 mmol) was treated with 40 mL of a methanolic solution ofNaOH 5% at reflux under magnetic stirring for 2 hrs. MeOH was thenremoved, the residue was dissolved in AcOEt (60 mL) and washed with H₂O(60 mL) and brine (60 mL). The organic layer was dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The oil residue wasdissolved in THF (40 mL) and treated with ethyl chloroformiate (0.57 mL,6.04 mmol) and triethylamine (0.90 mL, 6.47 mmol). The mixture wasvigorously stirred for 1 hr. Once the reaction was completed, themixture was diluted with AcOEt (60 mL), washed with H₂O (30 mL), HCl 1 N(30 mL), brine (30 mL) dried over anhydrous Na₂SO₄ and concentratedunder reduced pressure, to obtain the desired intermediate 3 inquantitative yield. The crude was used for the next step without furtherpurification.

3α,7α-dimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (4)

To a solution of 3 (2.01 g, 3.96 mmol) in DMF (40 mL), cyanuric chloride(0.48 g, 7.92 mmol) were added, and the reaction was stirred at roomtemperature for 16 hrs. The mixture was poured into AcOEt (100 mL),washed with H₂O (5×50 mL) and brine, (30 mL) dried over anhydrous Na₂SO₄and concentrated under reduced pressure. The residue was purified bysilica gel flash chromatography with petroleum ether/AcOEt as eluent(9:1→65:35, v/v), to obtain 1.43 g (2.93 mmol, 74%) of 4.

3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-24-N-hydroxy-cholanamidine (5)

To a solution of 4 (0.79 g, 1.61 mmol) in EtOH (45 mL), hydroxylaminechlorohydrate (1.68 g, 24.20 mmol) and sodium carbonate decahydrate(6.92 g, 24.20 mmol) were added and the mixture refluxed for 24 hrs.Volatiles were removed under reduced pressure and the resulting residuewas dissolved in EtOAc (50 mL), washed with H₂O (3×50 mL), brine (50mL), dried over anhydrous Na₂SO₄, filtered and concentrated underreduced pressure, to obtain 5 (0.81 g) in nearly quantitative yield. Thecrude was used as such for the next step.

3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-24-N[(ethoxycarbonyl)oxy]imidocholanamide (6)

To a solution of 5 (0.81 g, 1.61 mmol) in distilled CH₂Cl₂ (30 mL),cooled at 0° C. and under N₂ atmosphere, ethyl chloroformate (0.20 mL,2.10 mol) and pyridine (0.19 mL, 2.42 mmol) were added dropwise and thereaction mixture was stirred at room temperature for 1 hr. The reactionwas quenched with H₂O (15 mL) and the two phases were separated. Theorganic layer was thus washed with H₂O (3×15 mL), brine (15 mL), driedover anhydrous Na₂SO₄, filtered and concentrated under reduced pressureto afford 6 as crude (0.83 g), which was used for the next step withoutfurther purification.

3α,7α-Dimethoxymethyloxy-6α-ethyl-24-nor-5β-23([1,2,4]-oxadiazole-3-one-5yl)-cholane(7)

A solution of crude 6 (0.83 g) in toluene (15 mL) and pyridine (3 mL)was refluxed for 48 hrs. The mixture was diluted with AcOEt (30 mL),washed with H₂O (3×50 mL), brine (30 mL), dried over anhydrous Na₂SO₄,filtered and concentrated under reduced pressure, to yield 0.81 g of 7which was used as such for the next step.

3α,7α-Dihydroxy-6α-ethyl-24-nor-5β-23([1,2,4]-oxadiazole-3-one-5-yl)-cholane(Compound 16)

To a solution of crude 7 (0.81 g) in acetone (15 mL), HCl 3 N (5 mL) wasadded, and the mixture was stirred at 50° C. for 6 hrs. The organicsolvent was removed under reduced pressure, the residue was dissolved inCH₂Cl₂ (30 mL) and washed with H₂O (3×30 mL), brine (30 mL), dried overanhydrous Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was purified by silica gel flash chromatography using a solutionof CH₂Cl₂/MeOH/AcOH (97:3:0.1→93:7:0.1, v/v/v) as eluent to afford 0.27g (0.59 mmol, 36% from intermediate 4) of pure Compound 16.

rf: 0.49 (TLC: Silica Gel F₂₅₄S; eluent: CH₂Cl₂/MeOH/AcOH 90:10:1).¹H-NMR (CD₃OD, 400 MHz) δ: 0.70 (3H, s, 18-CH₃), 0.90-0.96 (6H, m,19-CH₃+CH₂CH₃), 1.02 (3H, d, J=6.1 Hz, 21-CH₃), 2.43-2.43 (1H, m,23-CH₂), 2.57-2.64 (1H, m, 23-CH₂), 3.29-3.33 (1H, m, 3-CH), 3.66 (1H,s, 7-CH). ¹³C-NMR (CDCl₃, 100.6 MHz) δ: 12.9, 13.1, 19.6, 22.9, 23.8,24.4, 24.6, 25.5, 30.2, 32.1, 33.9, 35.3, 35.4, 37.5, 37.6, 37.7, 41.9,42.5, 44.0, 44.7, 47.8, 52.6, 58.0, 72.0, 74.1, 162.7, 163.2.

Example 17: Synthesis of Compound 17

Methyl 3α,7α-dihydroxy-6α-ethyl-5-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-53-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL). The combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-Diacetoxy-6α-ethyl-23-oxo-24,24,24-trifluoromethyl-5β-cholane (6)

To a solution of 5 (14.20 g, 28.98 mmol) in toluene (125 mL) cooled at0° C., pyridine (11.44 g, 144.90 mmol) and trifluoroacetic anhydride(30.43 g, 144.90 mmol) were added. The mixture was refluxed for 18 hrs.After cooling at room temperature, the dark mixture was treated with H₂O(120 mL) at 45° C. for 1 hr, cooled at room temperature and acidified bythe careful addition of HCl 1 N (100 mL). The mixture was then extractedwith AcOEt (3×80 mL), the collected organic layers were washed withbrine (100 mL), dried over anhydrous Na₂SO₄, filtered under vacuum andconcentrated under reduced pressure. The brown oil residue was filteredthrough a silica gel pad (h: 10 cm, φ: 4 cm), collecting the crude withpetroleum ether/AcOEt (8:2, v/v) and obtaining the desiredtrifluoromethyl ketone 6 as pale yellow solid (15.7 g), which was usedfor the next step without further purification.

3α,7α-Diacetoxy-6α-ethyl-23-lactol derivative (7)

To a solution of crude 6 (15.7 g) in acetonitrile (415 mL) in a flaskequipped with mechanical stirring and repaired from light, ^(t)BuOH (135mL) and EDTA (170 mg, 0.584 mmol) dissolved in H₂O (395 mL) were added.NaHCO₃ (36.79 g, 438.00 mmol) and oxone (89.64 g, 146.00 mmol) wereadded portionwise, and the resulting suspension was vigorously stirredfor 18 hrs. The mixture was filtered to remove the solid, diluted withbrine (100 mL) and extracted with Et₂O (3×150 mL). The combined organiclayers were washed with brine (150 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was filtered through asilica gel pad (h: 12 cm, φ: 5 cm), collecting the crude with petroleumether/AcOEt (9:1, v/v). 9.60 g of desired lactol 7 were obtained. Thecrude material was used as such for the next step.

3α,7α-Diacetoxy-6α-ethyl-23-lactone derivative (8)

To a solution of 7 (9.60 g, 17.20 mmol) in MeOH (50 mL), a solution ofaqueous KOH 10 M (25.8 mL, 258.0 mmol) was added and the mixture wasstirred at reflux for 18 hrs. MeOH was removed under reduced pressure,H₂O (25 mL) was added and the resulting mixture was refluxed foradditional 24 hrs. After cooling at room temperature, the mixture waswashed with Et₂O (3×50 mL), acidified with HCl 3 N and extracted withCHCl₃ (3×150 mL). The collected organic layers were dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby silica gel flash chromatography eluting with an isocratic solution ofCHCl₃/MeOH/AcOH (97:3:0.1, v/v). After removal of solvent, 5.70 g (mmol,48% from intermediate 5) of desired intermediate 8 were obtained.

3α,7α-Dimethoxymethyloxy-6α-ethyl-23-lactone derivative (9)

To a solution of lactone 8 (1.75 g, 4.33 mmol) in CH₂Cl₂ (30 mL),diisopropylethylamine (5.03 g, 38.98 mmol), dimethylaminopyridine (0.05g, 0.43 mmol) and chloromethyl methyl ether (2.08 g, 25.99 mmol) weresequentially added, and the mixture was refluxed for 48 hrs. Thereaction was quenched by adding H₂O (30 mL) and the two phases wereseparated. The organic phase was washed with HCl 1 N (30 mL), with asaturated solution of NaHCO₃ (30 mL), brine (50 mL), dried overanhydrous Na₂SO₄, filtered under vacuum and concentrated under reducedpressure. The protected derivative 9 was used for the following stepwithout further purification.

3α,7α-Dimethoxymethyloxy-6α-ethyl-163,23-dihydroxy-24-nor-5β-cholane(10) To a suspension of LiAlH₄ (0.49 g, 12.99 mmol) in THF (30 mL)cooled at 0° C., a solution of 9 (2.13 g, 4.33 mmol) in THF (20 mL) wasadded dropwise. The reaction was stirred for 30′. Na₂SO₄ decahydrate wasslowly and cautiously added portionwise, until the hydrogen liberationdisappeared. The mixture was filtered under vacuum washing the solidresidue with AcOEt (5×5 mL); the collected organic phases wereconcentrated under reduced pressure, to afford 1.91 g (3.86 mmol, 89%)of the desired tetrahydroxy bile derivative 10 which was for the nextstep without further purification.

3α,7α-Dimethoxymethyloxy-6α-ethyl-16β-hydroxy-23-acetoxy-24-nor-5β-cholane(11)

To a solution of 10 (1.42 g, 2.86 mmol) in CH₂Cl₂ (120 mL), Ac₂O (0.81mL, 8.59 mmol) and Et₃N (1.81 mL, 12.88 mmol) were added, and theresulting solution was stirred at room temperature for 12 hrs. Themixture was poured into a saturated solution of NaHCO₃ (100 mL) andextracted with CH₂Cl₂ (2×60 mL). The combined organic layers were washedwith H₂O (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude 11 (1.46 g) was used assuch for the next step.

3α,7α,16β-Trimethoxymethyloxy 6α-ethyl-23-acetoxy-24-nor-5β-cholane (12)

To a solution of 11 (1.46 g, about 2.86 mmol) in CH₂Cl₂ (50 mL),diisopropylethylamine (1.97 mL, 11.45 mmol), dimethylaminopyridine (0.03g, 0.27 mmol) and chloromethyl methyl ether (0.65 mL, 8.59 mmol) weresequentially added. The mixture was refluxed for 5 hrs. The reaction wasquenched by adding H₂O (30 mL) and the two phases were separated. Theorganic phase was washed with HCl 1 N (30 mL), with a saturated solutionof NaHCO₃ (30 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The derivative 12 (1.51 g) was usedfor the following step without further purification.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-hydroxy-24-nor-5β-cholane (13)

To a solution of 12 (1.51 g, about 2.86 mmol) in MeOH (50 mL), NaOH(0.57 g, 14.31 mmol) was added and the mixture was refluxed for 3 hrs.The reaction was cooled at room temperature and the solvent was removedunder reduced pressure. The crude was dissolved in CH₂Cl₂ (50 mL),washed with H₂O (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The residue was purified by silicagel flash chromatography eluting with ethyl acetate in petroleum ether(from 5 to 30%) obtaining the desired compound 13 (1.35 g, 2.49 mmol,87% from intermediate 10) as pale yellow oil.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-iodio-24-nor-5β-cholane (14)

To a solution of triphenylphosphine (4.6 g, 17.56 mmol) in CH₂Cl₂ (50mL), iodine (2.05 g, 16.18 mmol) was added. After 10′, imidazole (1.16g, 17.10 mmol) was added to the solution. After additional 15′, asolution of alcohol 13 (1.25 g, 2.31 mmol) in CH₂Cl₂ (50 mL) was addedand the resulting mixture was stirred at room temperature for 48 hrs.The reaction was then poured into H₂O (100 mL), the phases wereseparated and the aqueous phase was extracted with CH₂Cl₂ (2×60 mL). Thecombined organic layers were washed with brine (100 mL), dried overanhydrous Na₂SO₄ and evaporated under reduced pressure. The crude waspurified by silica gel flash chromatography eluting with ethyl acetatein petroleum ether (from 5 to 20%) yielding 1.05 g (1.65 mmol, 71%) ofthe desired pure iodo derivative 14.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (15)

To a solution of iodo derivative 14 (1.03 g, 1.58 mmol) in DMSO (15 mL),sodium cyanide (0.09 g, 1.90 mmol) was added and the mixture was stirredat 80° C. for 3 hrs. The mixture was then allowed to cool to roomtemperature, diluted with CH₂Cl₂ (100 mL), washed with a saturatedsolution of NaHCO₃ (50 mL), H₂O (50 mL), brine (50 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The mixturewas purified by silica gel flash chromatography eluting with ethylacetate in petroleum ether (from 10 to 30%) to give 0.80 g (1.45 mmol,92%) of pure 15.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-[1-(tributylstannyl)-tetrazol-5-yl]-24-nor-5β-cholane(16)

A solution of nitrile 15 (0.68 g, 1.14 mmol) in toluene (12 mL) wasrefluxed with azidotributyltin(IV) (1.91 g, 5.72 mmol) for 36 hrs. Themixture was cooled at room temperature, diluted with EtOAc (15 mL),washed with H₂O (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue (0.82 g) was used forthe following step without further purification.

3α,7α,16β-Trihydroxy-6α-ethyl-23-(tetrazol-5-yl)-24-nor-5β-cholane(Compound 17)

To a solution of crude 16 (0.80 g) in MeOH (20 mL), HCl 3 N (5 mL) wasadded and the mixture was stirred at 50° C. for 48 hrs. The mixture wascooled at room temperature and treated with then NaOH 3 N (7 mL). Afterevaporation of the solvent, the crude residue was dissolved into H₂O (50mL) and washed with Et₂O (3×40 mL). The aqueous phase was then acidifiedup to pH=1 with HCl 3 N and extracted with a mixture of EtOAc/MeOH (9:1,v/v, 3×50 mL). The combined organic layers were washed with brine (100mL), dried over anhydrous Na₂SO₄ and concentrated under reducedpressure. The mixture was purified by silica gel flash chromatographyeluting with methanol in chloroform (from 1 to 10%) in the presence of0.1% of AcOH. 0.28 g of the final compound Compound 17 were obtained aswhite solid (54% from intermediate 15).

rf: 0.53 (TLC: Silica Gel 60 F₂₅₄S; eluent: CHCl₃/MeOH/AcOH 90:10:1).¹H-NMR (CD₃OD, 400 MHz) δ: 0.87-0.93 (9H, m, 18-CH₃+19-CH₃+CH₂CH₃), 1.07(3H, d, J=6.2 Hz, 21-CH₃), 2.12-2.17 (1H, m, 22-CH₂), 2.37-2.41 (1H, m,22-CH₂), 2.91-3.08 (2H, m, 23-CH₂), 3.31-3.35 (1H, m, 3-CH), 3.66 (1H,s, 7-CH), 4.40-4.44 (1H, m, 16-CH). ¹³C-NMR (CDCl₃, 100.6 MHz) δ: 10.5,11.8, 16.9, 19.9, 20.1, 22.0, 22.2, 29.5, 29.7, 32.7, 32.8, 33.0, 35.1(×2), 35.4, 39.5, 39.6, 41.6, 42.0, 45.4, 48.0, 61.1, 69.5, 71.6 (×2),157.1.

Example 18: Synthesis of Compound 18

Methyl 3α,7α-dihydroxy-6α-ethyl-5-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-53-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL), the combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-Diacetoxy-6α-ethyl-23-oxo-24,24,24-trifluoromethyl-5β-cholane (6)

To a solution of 5 (14.20 g, 28.98 mmol) in toluene (125 mL) cooled at0° C., pyridine (11.44 g, 144.90 mmol) and trifluoroacetic anhydride(30.43 g, 144.90 mmol) were added. The mixture was refluxed for 18 hrs.After cooling at room temperature, the dark mixture was treated with H₂O(120 mL) at 45° C. for 1 hr, cooled at room temperature and acidified bythe careful addition of HCl 1 N (100 mL). The mixture was then extractedwith AcOEt (3×80 mL), the collected organic layers were washed withbrine (100 mL), dried over anhydrous Na₂SO₄, filtered under vacuum andconcentrated under reduced pressure. The brown oil residue was filteredthrough a silica gel pad (h: 10 cm, φ: 4 cm), collecting the crude withpetroleum ether/AcOEt (8:2, v/v) and obtaining the desiredtrifluoromethyl ketone 6 as pale yellow solid (15.7 g), which was usedfor the next step without further purification.

3α,7α-Diacetoxy-6α-ethyl-23-lactol derivative (7)

To a solution of crude 6 (15.7 g) in acetonitrile (415 mL) in a flaskequipped with mechanical stirring and repaired from light, ^(t)BuOH (135mL) and EDTA (170 mg, 0.584 mmol) dissolved in H₂O (395 mL) were added.NaHCO₃ (36.79 g, 438.00 mmol) and oxone (89.64 g, 146.00 mmol) wereadded portionwise, and the resulting suspension was vigorously stirredfor 18 hrs. The mixture was filtered to remove the solid, diluted withbrine (100 mL) and extracted with Et₂O (3×150 mL). The combined organiclayers were washed with brine (150 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The residue was filtered through asilica gel pad (h: 12 cm, φ: 5 cm), collecting the crude with petroleumether/AcOEt (9:1, v/v). 9.60 g of desired lactol 7 were obtained. Thecrude material was used as such for the next step.

3α,7α-Diacetoxy-6α-ethyl-23-lactone derivative (8)

To a solution of 7 (9.60 g, 17.20 mmol) in MeOH (50 mL), a solution ofaqueous KOH 10 M (25.8 mL, 258.0 mmol) was added and the mixture wasstirred at reflux for 18 hrs. MeOH was removed under reduced pressure,H₂O (25 mL) was added and the resulting mixture was refluxed foradditional 24 hrs. After cooling at room temperature, the mixture waswashed with Et₂O (3×50 mL), acidified with HCl 3 N and extracted withCHCl₃ (3×150 mL). The collected organic layers were dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The residue was purifiedby silica gel flash chromatography eluting with an isocratic solution ofCHCl₃/MeOH/AcOH (97:3:0.1, v/v). After removal of solvent, 5.70 g (mmol,48% from intermediate 5) of desired intermediate 8 were obtained.

3α,7α-Dimethoxymethyloxy-6α-ethyl-23-lactone derivative (9)

To a solution of lactone 8 (1.75 g, 4.33 mmol) in CH₂Cl₂ (30 mL),diisopropylethylamine (5.03 g, 38.98 mmol), dimethylaminopyridine (0.05g, 0.43 mmol) and chloromethyl methyl ether (2.08 g, 25.99 mmol) weresequentially added, and the mixture was refluxed for 48 hrs. Thereaction was quenched by adding H₂O (30 mL) and the two phases wereseparated. The organic phase was washed with HCl 1 N (30 mL), with asaturated solution of NaHCO₃ (30 mL), brine (50 mL), dried overanhydrous Na₂SO₄, filtered under vacuum and concentrated under reducedpressure. The protected derivative 9 was used for the following stepwithout further purification.

3α,7α-Dimethoxymethyloxy-6α-ethyl-16β,23-dihydroxy-24-nor-5β-cholane(10)

To a suspension of LiAlH₄ (0.49 g, 12.99 mmol) in THF (30 mL) cooled at0° C., a solution of 9 (2.13 g, 4.33 mmol) in THF (20 mL) was addeddropwise. The reaction was stirred for 30′. Na₂SO₄ decahydrate wasslowly and cautiously added portionwise, until the hydrogen liberationdisappeared. The mixture was filtered under vacuum washing the solidresidue with AcOEt (5×5 mL); the collected organic phases wereconcentrated under reduced pressure, to afford 1.91 g (3.86 mmol, 89%)of the desired tetrahydroxy bile derivative 10 which was for the nextstep without further purification.

3α,7α-Dimethoxymethyloxy-6α-ethyl-16β-hydroxy-23-acetoxy-24-nor-5β-cholane(11)

To a solution of 10 (1.42 g, 2.86 mmol) in CH₂Cl₂ (120 mL), Ac₂O (0.81mL, 8.59 mmol) and Et₃N (1.81 mL, 12.88 mmol) were added, and theresulting solution was stirred at room temperature for 12 hrs. Themixture was poured into a saturated solution of NaHCO₃ (100 mL) andextracted with CH₂Cl₂ (2×60 mL). The combined organic layers were washedwith H₂O (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The crude 11 (1.46 g) was used assuch for the next step.

3α,7α,16β-Trimethoxymethyloxy 6α-ethyl-23-acetoxy-24-nor-5β-cholane (12)

To a solution of 11 (1.46 g, about 2.86 mmol) in CH₂Cl₂ (50 mL),diisopropylethylamine (1.97 mL, 11.45 mmol), dimethylaminopyridine (0.03g, 0.27 mmol) and chloromethyl methyl ether (0.65 mL, 8.59 mmol) weresequentially added. The mixture was refluxed for 5 hrs. The reaction wasquenched by adding H₂O (30 mL) and the two phases were separated. Theorganic phase was washed with HCl 1 N (30 mL), with a saturated solutionof NaHCO₃ (30 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andconcentrated under reduced pressure. The derivative 12 (1.51 g) was usedfor the following step without further purification.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-hydroxy-24-nor-5β-cholane (13)

To a solution of 12 (1.51 g, about 2.86 mmol) in MeOH (50 mL), NaOH(0.57 g, 14.31 mmol) was added and the mixture was refluxed for 3 hrs.The reaction was cooled at room temperature and the solvent was removedunder reduced pressure. The crude was dissolved in CH₂Cl₂ (50 mL),washed with H₂O (50 mL), brine (50 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The residue was purified by silicagel flash chromatography eluting with ethyl acetate in petroleum ether(from 5 to 30%) obtaining the desired compound 64 (1.35 g, 2.49 mmol,87% from intermediate 10) as pale yellow oil.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-iodio-24-nor-5β-cholane (14)

To a solution of triphenylphosphine (4.6 g, 17.56 mmol) in CH₂Cl₂ (50mL), iodine (2.05 g, 16.18 mmol) was added. After 10′, imidazole (1.16g, 17.10 mmol) was added to the solution. After additional 15′, asolution of alcohol 13 (1.25 g, 2.31 mmol) in CH₂Cl₂ (50 mL) was addedand the resulting mixture was stirred at room temperature for 48 hrs.The reaction was then poured into H₂O (100 mL), the phases wereseparated and the aqueous phase was extracted with CH₂Cl₂ (2×60 mL). Thecombined organic layers were washed with brine (100 mL), dried overanhydrous Na₂SO₄ and evaporated under reduced pressure. The crude waspurified by silica gel flash chromatography eluting with ethyl acetatein petroleum ether (from 5 to 20%) yielding 1.05 g (1.65 mmol, 71%) ofthe desired pure iodo derivative 14.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-23-cyano-24-nor-5β-cholane (15)

To a solution of iodo derivative 14 (1.03 g, 1.58 mmol) in DMSO (15 mL),sodium cyanide (0.09 g, 1.90 mmol) was added and the mixture was stirredat 80° C. for 3 hrs. The mixture was then allowed to cool to roomtemperature, diluted with CH₂Cl₂ (100 mL), washed with a saturatedsolution of NaHCO₃ (50 mL), H₂O (50 mL), brine (50 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The mixturewas purified by silica gel flash chromatography eluting with ethylacetate in petroleum ether (from 10 to 30%) to give 0.80 g (1.45 mmol,92%) of pure 15.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-5β-24-N-hydroxy-cholanamidine(16)

To a solution of 15 (0.16 g, 0.29 mmol) in ethanol (15 mL), sodiumcarbonate decahydrate (1.25 g, 4.36 mmol) and hydroxylaminehydrochloride (0.30 g, 4.36 mmol) were added. The resulting mixture wasrefluxed for 18 hrs. The solvent was removed under reduced pressure, thecrude was dissolved in CH₂Cl₂ (30 mL), washed with H₂O (30 mL), brine(30 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The residue (0.17 g) was used as such for the following step.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-5β-24-N[(ethoxycarbonyl)oxy]imidocholanamide (17)

To a solution of crude hydroxyamidine 16 (0.17 g, 0.29 mmol) in CH₂Cl₂(10 mL), ethylchloroformate (0.04 g, 0.38 mmol) and pyridine (0.03 g,0.44 mmol) were added at 0° C. The mixture was stirred at roomtemperature for 1 hr. The reaction was quenched by adding H₂O (15 mL).The phases were separated and the water phase was extracted with CH₂Cl₂(2×15 mL). The combined organic layers were washed with brine (30 mL),dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. Thecrude residue (0.18 g) was used for the following step without furtherpurification.

3α,7α,16β-Trimethoxymethyloxy-6α-ethyl-24-nor-5β-23-([1,2,4]-oxadiazole-3-one-5yl)-cholane(18)

A solution of crude 17 (0.18 g, 0.29 mmol) in toluene (5 mL) wasrefluxed in the presence of pyridine (1 mL) for 48 hrs. The reaction wasdiluted with EtOAc (20 mL), washed with H₂O (30 mL) and HCl 3 N (30 mL),with a saturated solution of NaHCO₃ (30 mL), brine (50 mL), dried overanhydrous Na₂SO₄ and concentrated under reduced pressure. The derivative18 (0.17 g) was used for the following step without furtherpurification.

3α,7α,16β-Trihydroxy-6α-ethyl-24-nor-5β-23-([1,2,4]-oxadiazole-3-one-5yl)-cholane(Compound 18)

To a solution of crude compound 18 (0.19 g) in acetone (5 mL), HCl 3 N(1 mL) was added and the mixture was stirred at 35° C. for 48 hrs. Thesolvent was evaporated under reduced pressure, suspended in H₂O (10 mL)and extracted with CH₂Cl₂ (2×10 mL). The combined organic layers werewashed with H₂O (10 mL), brine (10 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The mixture was purified by silicagel flash chromatography using a solution of methanol in chloroform(from 1 to 10%) in the presence of 0.1% of AcOH. Evaporation of thesolvent afforded 11 mg of Compound 18 (8% yield from 13) as white solid.

rf: 0.51 (TLC: Silica Gel 60 F₂₅₄S; eluent: CHCl₃/MeOH/AcOH 90:10:1).¹H-NMR (CD₃OD, 400 MHz) δ: 0.87-0.93 (9H, m, 18-CH₃+19-CH₃+CH₂CH₃), 1.03(3H, d, J=6.4 Hz, 21-CH₃), 2.36-2.32 (1H, m, 22-CH₂), 2.50-2.57 (1H, m,23-CH₂), 2.63-2.67 (1H, m, 23-CH₂), 3.25-3.33 (1H, m, 3-CH), 3.65 (1H,s, 7-CH), 4.33-4.37 (1H, m, 16-CH). ¹³C-NMR (CDCl₃, 100.6 MHz) δ: 10.9,12.2, 17.2, 20.5, 22.1, 22.4, 22.6, 30.0, 30.1, 31.5, 33.2, 33.4, 35.5(×2), 36.0, 39.9, 40.0, 42.0, 42.5, 45.8, 61.3, 70.0, 71.8, 72.0, 160.8,161.1.

Example 19: Synthesis of Compound 19

Methyl 3α,7α-dihydroxy-6-ethyl-5β-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-5β-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL). The combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5β-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-dihydroxy-6α-ethyl-24-nor 5β-cholan-23-oic acid (6)

To a solution of 3α,7α-diacetoxy-6α-ethyl-24-nor-51-cholanoic acid (5)(5.27 g, 10.75 mmol) in MeOH (70 mL), an aqueous solution of KOH (6.02g, 107.5 mmol in 10 mL of H₂O) was added. The reaction was divided in 6batches of about 15 mL. Each lot was submitted to microwave irradiation(T=120° C., P_(max)=270 psi, Power_(max)=200 W) for 2 hrs. The diverselots were collected, the solvent was removed under reduced pressure, thecrude was dissolved in H₂O (100 mL) and extracted with Et₂O (2×50 mL).The aqueous phase was acidified with HCl 3 N and extracted with CH₂Cl₂(3×80 mL). The combined organic layers were washed with H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The crude was purified by flash chromatography eluting withMeOH in CHCl₃ (from 0 to 10%) in the presence of 0.1% of AcOH to furnishthe desired acid 6 as white solid (3.85 g, 9.46 mmol, 88%).

Methyl 3α,7α-dihydroxy-6α-ethyl-24-nor 5β-cholan-23-oate (7)

To a solution of 3α,7α-dihydroxy-6α-ethyl-24-nor 51-cholan-23-oic acid(6) (0.80 g, 1.72 mmol) in MeOH (20 mL) p-toluensulfonic acidmonohydrate (0.04 g, 0.17 mmol) was added and the mixture was sonicatedat 25° C. for 4 hrs. The solvent was removed under reduced pressure, theresidue was dissolved in CHCl₃ (80 mL), washed with a saturated solutionof NaHCO₃ (80 mL), H₂O (80 mL), brine (80 mL), dried over anhydrousNa₂SO₄ and evaporated under reduced pressure. The white solid thusobtained (0.82 g, 1.72 mmol) was used for the following step withoutfurther purification.

3α,7α-Dihydroxy-6α-ethyl-N-(2-hydroxyethyl)-24-nor-5β-cholan-23-amide(Compound 19)

A mixture of methyl ester 7 (0.82 g, 1.72 mmol) and ethanolamine (8.08g, 132.24 mmol) in MeOH (8 mL) was submitted to microwave irradiation(T=130° C., P_(max)=200 psi, Power_(max)=200 W) for 1 hr. The mixturewas concentrated under reduced pressure, the residue was dissolved inCH₂Cl₂ (50 mL), washed with HCl 3 N (50 mL), H₂O (50 mL), brine (50 mL),dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. Thecrude was purified by silica gel flash chromatography eluting withCHCl₃/MeOH (0→10%+0.1% of AcOH) to furnish the desired derivativeCompound 19 as white solid (0.60 g, 1.34 mmol, 78%).

rf: 0.42 (TLC: Silica Gel F₂₅₄S; eluent: CHCl₃/MeOH/AcOH 90:10:0.1).¹H-NMR (DMSO-d6, 400 MHz) δ 0.53 (3H, s, 18-CH₃), 0.71-0.77 (9H, m,19-CH₃+CH₂CH₃+21-CH₃), 2.05 (1H, m, 22-CH₂), 2.98-3.04 (3H, m,3-CH+CH₂CH₂OH), 3.27 (2H, t, J=6.0 Hz, CH₂CH₂OH), 3.39 (1H, s, 7-CH),3.10-3.40 (1H, bs, OH), 3.96 (1H, s, OH), 4.05-4.37 (1H, bs, OH).¹³C-NMR (CD₃OD, 400 MHz) δ 12.1 (×2), 19.4, 20.7, 22.5, 23.4 (×2), 28.2,30.7, 32.9, 33.8, 33.9, 35.5, 35.8, 41.6, 41.7, 42.4, 43.2, 45.6, 50.5,56.4, 60.3, 68.7, 70.9, 172.3.

Example 20: Synthesis of Compound 20

Methyl 3α,7α-dihydroxy-6α-ethyl-5-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-5β-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL), the combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-dihydroxy-6α-ethyl-24-nor 5β-cholan-23-oic acid (6)

To a solution of 3,7α-diacetoxy-6α-ethyl-24-nor-51-cholanoic acid (5)(5.27 g, 10.75 mmol) in MeOH (70 mL), an aqueous solution of KOH (6.02g, 107.5 mmol in 10 mL of H₂O) was added. The reaction was divided in 6batches of about 15 mL. Each lot was submitted to microwave irradiation(T=120° C., P_(max)=270 psi, Power_(max)=200 W) for 2 hrs. The diverselots were collected, the solvent was removed under reduced pressure, thecrude was dissolved in H₂O (100 mL) and extracted with Et₂O (2×50 mL).The aqueous phase was acidified with HCl 3 N and extracted with CH₂Cl₂(3×80 mL). The combined organic layers were washed with H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The crude was purified by flash chromatography eluting withMeOH in CHCl₃ (from 0 to 10%) in the presence of 0.1% of AcOH to furnishthe desired acid 6 as white solid (3.85 g, 9.46 mmol, 88%).

3α,7α-Dihydroxy-6α-ethyl-N-(2-hydroxyethyl)-24-nor5β-cholan-23-hydroxyamide (Compound 20)

To a solution of 6α-ethyl-3α,7α-dihydroxy-23-nor-5β-cholanoate (6) (0.50g, 1.23 mmol) in dry DMF (20 mL), DMT-MM (1.36 g, 4.92 mmol) andtriethylamine (1.24 g, 12.30 mmol) were added and the mixture wasstirred at room temperature for 1 hr. Freshly prepared solution of NH₂OH(0.06 g, 1.84 mmol) in dry DMF was added and the mixture was refluxedfor 4 hrs. The reaction was poured into H₂O (40 mL) and extracted withCHCl₃ (3×30 mL). The combined organic layers were washed with HCl 1 N(40 mL), with a saturated solution of NaHCO₃ (40 mL), H₂O (40 mL), brine(40 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The crude was purified by silica gel flash chromatographyusing a solution of CHCl₃/MeOH (0→8%+0.1% AcOH). The desired compoundCompound 20 was obtained as white solid (0.24 g, 0.57 mmol, 46%).

rf: 0.24 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 50:50). ¹H-NMR(CD₃OD, 400 MHz) δ 0.61 (3H, s, 18-CH₃), 0.77-0.88 (9H, m,19-CH₃+CH₂CH₃+21-CH₃), 2.13-2.16 (1H, m, 22-CH₂), 3.18-3.20 (1H, m,3-CH), 3.53 (1H, s, 7-CH). ¹³C-NMR (CD₃OD, 400 MHz) δ 10.59, 10.81,17.9, 20.5, 22.0, 22.3, 23.0, 27.8, 29.9, 32.9, 33.0, 33.6, 35.1, 35.2,39.5 (×2), 40.0, 41.6, 42.1, 45.2, 50.2, 56.3, 69.5, 71.6, 170.9.

Example 21: Synthesis of Compound 21

Methyl 3α,7α-dihydroxy-6α-ethyl-5β-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-53-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL), the combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the obtained residue wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), a saturatedsolution of NaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄and evaporated under reduced pressure. The biphenyl derivative 3 wasused for the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5β-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2 N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-dihydroxy-6α-ethyl-24-nor 5β-cholan-23-oic acid (6)

To a solution of 3α,7α-diacetoxy-6α-ethyl-24-nor-5β-cholanoic acid (5)(5.27 g, 10.75 mmol) in MeOH (70 mL), an aqueous solution of KOH (6.02g, 107.5 mmol in 10 mL of H₂O) was added. The reaction was divided in 6batches of about 15 mL. Each lot was submitted to microwave irradiation(T=120° C., P_(max)=270 psi, Power_(max)=200 W) for 2 hrs. The diverselots were collected, the solvent was removed under reduced pressure, thecrude was dissolved in H₂O (100 mL) and extracted with Et₂O (2×50 mL).The aqueous phase was acidified with HCl 3 N and extracted with CH₂Cl₂(3×80 mL). The combined organic layers were washed with H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The crude was purified by flash chromatography eluting withMeOH in CHCl₃ (from 0 to 10%) in the presence of 0.1% of AcOH to furnishthe desired acid 6 as white solid (3.85 g, 9.46 mmol, 88%).

Sodium 2-(3α,7α-dihydroxy-6α-ethyl-24-nor 5β-cholan-23-amido)-ethylsulfate (Compound 21)

To a solution of 6α-ethyl-3α,7α-dihydroxy-23-nor-5β-cholanoate (6) (0.90g, 2.21 mmol) in ethanol (25 mL), triethylamine (2.24 g, 22.13 mmol) and2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (1.37 g, 5.53 mmol) wereadded and the resulting mixture was stirred at room temperature for 30′at 50° C. for 1 hr. Sodium ethanolamine sulphate (prepared by reactionof ethanolamine and piridine sulfurtrioxide complex in acetonitrile in87% yield) (0.72 g, 4.43 mmol) was added to the mixture which wasreacted at 90° C. for 6 hrs. The mixture was concentrated under reducedpressure, the residue was dissolved in aqueous NaOH (5% in H₂O, 30 mL)and stirred for 30′. The aqueous phase was extracted with EtOAc (3×50mL) and concentrated under reduced pressure. The crude was purified byreverse phase flash chromatography eluting with acetonitrile in water(from 5 to 30%) affording 0.78 g (1.41 mmol, 64%) of Compound 21.

rf: 0.44 (TLC: Silica Gel 60 RP-8 F₂₅₄S; eluent: H₂O/MeCN 65:35). ¹H-NMR(CD₃OD, 400 MHz) δ 0.65 (3H, s, 18-CH₃), 0.80-0.84 (6H, m,19-CH₃+CH₂CH₃), 0.89 (3H, d, J=6.1 Hz, 21-CH₃), 3.28 (1H, dd, J₁=2.4 Hz,J₂=12.4 Hz, 22-CH₂), 3.20-3.25 (1H, m, 3-CH), 3.37 (2H, t, J=5.2 Hz,CH₂CH₂O), 3.57 (1H, s, 7-CH), 3.96 (2H, t, J=5.2 Hz, CH₂CH₂O). ¹³C-NMR(CD₃OD, 400 MHz) δ 10.5, 10.8, 18.3, 20.4, 22.0, 22.2, 23.0, 27.8, 29.7,33.0, 33.9, 35.1, 35.2, 38.6, 39.5, 40.0, 41.6, 42.3, 43.0, 45.4, 50.2,56.4, 65.5, 69.9, 71.7, 174.7.

Example 22: Synthesis of Compound 22

Methyl 3α,7α-dihydroxy-6α-ethyl-5-cholanoate (2)

To a solution of OCA (1) (5.0 g, 11.9 mmol) in MeOH (100 mL)p-toluensulfonic acid monohydrate (0.23 g, 1.19 mmol) was added and themixture was sonicated at room temperature for 90′. The solvent wasremoved under reduced pressure, the residue was dissolved in CHCl₃ (100mL), washed with a saturated solution of NaHCO₃ (100 mL), H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated under reducedpressure. The white solid thus obtained (5.17 g, 11.89 mmol) was usedfor the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (3)

To a solution of methyl 6α-ethyl-3α,7α-dihydroxy-5β-cholanoate (2) (5.17g, 11.89 mmol) in dry THF (125 mL), phenylmagnesium bromide 3 M in Et₂O(39.6 mL, 118.9 mmol) was added dropwise. The mixture was refluxed for12 hrs. After cooling at room temperature, the mixture was treated withH₂O (100 mL) and HCl 3 M (100 mL). The mixture was extracted with EtOAc(3×80 mL). The combined organic layers were washed with brine, driedover anhydrous Na₂SO₄ and evaporated under reduced pressure. The crudewas dissolved in MeOH (100 mL) and refluxed in the presence of HCl 37%(10 mL) for 1 hr. MeOH was evaporated, the residue obtained wasdissolved in EtOAc (120 mL), washed with H₂O (2×100 mL), saturatedNaHCO₃ (100 mL), brine (100 mL), dried over anhydrous Na₂SO₄ andevaporated under reduced pressure. The biphenyl derivative 3 was usedfor the next step without purification.

3α,7α-Diacetoxy-6α-ethyl-24,24-biphenyl-5β-cholan-23-ene (4)

To a solution of 3 (6.42 g, 11.89 mmol) in CH₂Cl₂ (70 mL), aceticanhydride (6.06 g, 59.45 mmol) and bismuth trifluoromethanesulfonate(0.39 g, 0.59 mmol) were added. The resulting mixture was stirred atroom temperature for 1 hr. A saturated aqueous solution of NaHCO₃ (50mL) was then carefully added and the phases were separated. The aqueouslayer was extracted with CH₂Cl₂ (2×50 mL). The combined organic layerswere washed with H₂O (100 mL), brine (100 mL), dried over anhydrousNa₂SO₄ and concentrated under reduced pressure. The crude was purifiedby silica gel flash chromatography using an eluent constituted bypetroleum ether/EtOAc (95:5→7:3, v/v) obtaining 5.56 g (8.91 mmol, 75%)of desired intermediate 4.

3α,7α-Diacetoxy-6α-ethyl-24-nor-5β-cholan-23-oic acid (5)

To a suspension of sodium periodate (21.13 g, 98.73 mmol) in H₂O (20mL), H₂SO₄ 2N in H₂O (3.22 mL) was added and the mixture was stirred atroom temperature for 1 hr. The mixture was cooled to 0° C. and treatedwith ruthenium trichloride hydrate (0.11 g, 0.55 mmol) which was addedin one portion. After 1 hr, acetonitrile (31 mL) was added to thesolution and after additional 5′, a solution of biphenyl derivative 4(6.85 g, 10.97 mmol) in EtOAc (43 mL) was added. The mixture was stirredat room temperature for 1 hr. The white solid thus formed was filteredoff, then the liquor was poured into H₂O (100 mL) and extracted withEtOAc (3×50 mL). The combined organic layers were filtered through aCelite pad, washed with a saturated solution of Na₂S₂O₃ in H₂O (100 mL),brine (100 mL), dried over anhydrous Na₂SO₄ and concentrated underreduced pressure. The residue was purified by silica gel flashchromatography eluting with EtOAc in petroleum ether from 10 to 50%. Thedesired acid 5 was obtained as white solid (5.27 g, 10.75 mmol, 98%).

3α,7α-Dihydroxy-6α-ethyl-24-nor 5β-cholan-23-oic acid (6)

To a solution of 3α,7α-diacetoxy-6α-ethyl-24-nor-5β-cholanoic acid (5)(5.27 g, 10.75 mmol) in MeOH (70 mL), an aqueous solution of KOH (6.02g, 107.5 mmol in 10 mL of H₂O) was added. The reaction was divided in 6batches of about 15 mL. Each lot was submitted to microwave irradiation(T=120° C., P_(max)=270 psi, Power_(max)=200 W) for 2 hrs. The diverselots were collected, the solvent was removed under reduced pressure, andthe crude was dissolved in H₂O (100 mL) and extracted with Et₂O (2×50mL). The aqueous phase was acidified with HCl 3 N and extracted withCH₂Cl₂ (3×80 mL). The combined organic layers were washed with H₂O (100mL), brine (100 mL), dried over anhydrous Na₂SO₄ and evaporated underreduced pressure. The crude was purified by flash chromatography elutingwith MeOH in CHCl₃ (from 0 to 10%) in the presence of 0.1% of AcOH tofurnish the desired acid 6 as white solid (3.85 g, 9.46 mmol, 88%).

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-24-nor-5β-cholan-23-oate (7)

To a solution of 3α,7α-dihydroxy-6α-ethyl-24-nor-5β-cholanoic acid (6)(3.0 g, 7.39 mmol) in MeOH (50 mL), p-toluensulfonic acid monohydrate(0.14 g, 0.74 mmol) was added, and the resulting mixture was sonicatedat room temperature for 4 hours. The solvent was removed under reducedpressure, the residue was dissolved in CHCl₃ (80 mL) and washed with asaturated solution of NaHCO₃ (50 mL), H₂O (50 mL) and brine (50 mL). Theorganic layer was dried over Na₂SO₄ and concentrated under reducedpressure. The ester thus obtained was dissolved in CH₂Cl₂ (30 mL) anddiisopropylethylamine (10.17 mL, 59.11 mmol),4-(N,N-dimethylamino)-pyridine (0.09 g, 0.74 mmol) andmethoxymethylchloride (3.35 mL, 44.33 mmol) were sequentially added tothe resulting solution. The mixture was stirred and refluxed for 24hours. The reaction then allowed to cool to room temperature and washedwith a saturated solution of NH₄Cl (30 mL), H₂O (30 mL) and brine (30mL). The organic layer was dried over Na₂SO₄ and concentrated underreduced pressure, to afford 3.57 g of 7 as pale yellow oil that was usedfor the following step without further purification (3.57 g, 7.02 mmol,95%).

Methyl3α,7α-dimethoxymethyloxy-6α-ethyl-22(R+S)-hydroxy-24-nor-5β-cholan-23-oate(9)

To a stirred solution of diisopropylamine (7.92 mL, 55.91 mmol) indistilled THF (30 mL) under N₂ atmosphere and cooled at −40° C., nBuLi(2.5 M in hexane, 21.52 mL, 53.81 mmol) was added dropwise. After 15minutes, the solution was cooled at −78° C. and chlorotrimethylsilane(7.28 mL, 57.30 mmol) was added dropwise. After additional 15 minutes, asolution of protected ester 7 (3.55 g, 6.99 mmol) in distilled THF (10mL) was added dropwise in about 20 minutes, maintaining the internaltemperature not over −70° C. Once the addition was finished, thereaction mixture was stirred at −78° C. for 1 additional hour and thenwas warmed at room temperature. Volatiles were removed under reducedpressure, and the residue was suspended in petroleum ether (80 mL) andfiltered under vacuum. The liquor was concentrated under reducedpressure to furnish the desiderate compound 9. The intermediate thusobtained was directly dissolved in distilled CH₂Cl₂ (20 mL) and addeddropwise to a 0° C. cooled suspension of freshly crystallized and aceticacid free lead(IV)tetraacetate (6.64 g, 10.484 mmol) in distilled CH₂Cl₂(30 mL) under N₂ atmosphere. The mixture was stirred at 0° C. for 30minutes then was filtered under vacuum through a Celite pad. Thefiltrate was concentrated under reduced pressure, and the residue wasquickly filtered through a silica gel pad (h: 8 cm, φ: 4 cm), collectingthe crude with petroleum ether/AcOEt (8:2, v/v). After evaporation ofthe solvents, the residue was dissolved in MeOH (30 mL) and to theresulting solution solid potassium carbonate (1.93 g, 13.98 mmol) wasadded. The resulting suspension was vigorously stirred at roomtemperature for 15 minutes. The mixture was then diluted with CH₂Cl₂ (40mL) and filtered under vacuum. The filtrate was further diluted withadditional CH₂Cl₂ (50 mL) and washed with brine (50 mL). The phases wereseparated, the aqueous phase was extracted with CH₂Cl₂ (3×40 mL), andall the collected organic layers were combined, dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was purified by flashchromatography by using petroleum ether/AcOEt from 80:20 (v/v) to 50:50(v/v) to afford 9 as mixture of two C22-epimers (1.17 g, 2.24 mmol,32%).

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-22-oxo-24-nor-5β-cholan-23-oate(10)

To a solution of oxalyl chloride (0.47 mL, 5.50 mmol) in distilledCH₂Cl₂ (15 mL) under N₂ atmosphere and cooled ad −60° C.,dimethylsulfoxide (0.78 mL, 10.99 mmol) diluted in CH₂Cl₂ (3 mL) wasadded dropwise. After 15 minutes, a solution of 22-hydroxy derivative 9(1.15 g, 2.20 mmol) in CH₂Cl₂ (15 mL) was added dropwise, and theresulting mixture was stirred at −60° C. for 1 hours. Then triethylamine(3.08 mL, 21.99 mmol) was added dropwise, and the mixture was slowlywarmed at room temperature. The reaction mixture was treated with KOH 1M(20 mL) for 5 minutes, the two phases were separated and the aqueous onewas extracted with CH₂Cl₂ (2×20 mL). The collected organic layers werewashed with brine (50 mL), dried over Na₂SO₄ and concentrated underreduced pressure. The residue was purified by flash chromatography,collecting the desired 22-oxo derivative 10 (0.88 g, 1.69 mmol, 77%) byusing petroleum ether/AcOEt from 90:10 (v/v) to 80:20 (v/v).

Methyl3,7-dimethoxymethyloxy-6α-ethyl-22,22-difluoro-24-nor-5β-cholan-23-oate(11)

To a solution of 22-keto derivative 10 (0.50 g, 0.96 mmol) in distilledTHF (9 mL) under N₂ atmosphere, bis(2-methoxyethyl)aminosulfurtrifluoride (Deoxo-Fluor® 50% in THF 3.29 mL, 7.67 mmol) was added andthe reaction was stirred at 50° C. for 16 hours. SupplementaryDeoxo-Fluor® (2.27 mL, 5.27 mmol) was added and the mixture was refluxedfor further 72 hours. The reaction was then allowed to cool to roomtemperature and the mixture was cautiously poured in a saturatedsolution of NaHCO₃ (40 mL) placed in a water-ice bath and under magneticstirring. Once the CO₂ release was finished, the mixture was extractedwith AcOEt (2×40 mL), the combined organic layers were washed with H₂O(60 mL), brine (60 mL), dried over Na₂SO₄ and concentrated under reducedpressure. The residue was filtered through a silica pad eluting withpetroleum ether/AcOEt 80:20 (v/v) and the crude compound 11 was used forthe following step without further purification.

3α,7α-Dihydroxy-6α-ethyl-22,22-difluoro-24-nor-5β-cholan-23-oic acid(Compound 22)

To a solution of derivative 11 (0.96 mmol) in MeOH (12 mL), HCl 37%(0.80 mL, 9.60 mmol) was added, and the mixture was stirred at 45° C.for 12 hours. Then sodium hydroxide (0.57 g, 14.39 mmol) was added, andthe mixture was stirred at 45° C. for additional 4 hours. The solventwas removed under reduced pressure, the residue was dissolved in H₂O (25mL) and washed with Et₂O (2×20 mL). The aqueous phase was acidified upto pH=5 by adding HCl 3N and extracted with AcOEt (3×30 mL). The solventwas removed under reduced pressure and the residue was purified by RP-18medium pressure liquid chromatography, by using H₂O/MeCN from 95:5 (v/v)to 40:60 (v/v) obtaining 0.06 g of Compound 22 as white solid (0.06 g,0.13 mmol, 14%)

Rf=0.55 (RP-C8 SiO₂, F-254s, H₂O/MeCN 60:40). M.p.=254-256° C. ¹H-NMR(DMSO-d₆, 400 MHz) δ: 0.60 (3H, s, 18-CH₃), 0.78-0.80 (6H, m,19-CH₃+CH₂CH₃), 0.92 (3H, d, J=6.6 Hz, 21-CH₃), 2.09-2.14 (1H, m,20-CH), 3.08-3.12 (1H, m, 3-CH), 3.47 (1H, s, 7-CH). ¹³C-NMR (DMSO-d₆,100.6 MHz) δ 11.8, 12.1, 20.9, 22.6, 23.5, 23.9, 27.8, 30.8, 31.1, 33.0,33.9, 35.6, 35.9, 41.6, 43.2, 45.7, 49.8, 50.8, 68.8, 71.0, 121.0(J_(C-F)=277.6 Hz), 167.2 (J_(C-F)=28.2 Hz).

Example 23: Synthesis of Compound 23

Methoxymethyl 3α,7α-dimethoxymethyloxy-6α-ethyl-5β-cholan-24-oate (2)

To a solution of OCA (1) (1.0 g, 2.38 mmol) in CH₂Cl₂ (40 mL),diisopropylethylamine (4.94 mL, 28.54 mmol), methoxymethylchloride (1.45mL, 19.03 mmol), and 4-(N,N-dimethylamino)-pyridine (0.06 g, 0.47 mmol)were sequentially added. The resulting mixture was stirred and refluxedfor 18 hours. The reaction was then washed with a saturated solution ofNH₄Cl (40 mL), H₂O (40 mL) and brine (40 mL). The organic layer wasdried over Na₂SO₄ and concentrated under reduced pressure to afford 1.18g of 2 as pale yellow oil that was used for the following step withoutfurther purification (1.18 g, 2.14 mmol).

2-(3α,7α-Dimethoxymethyloxy-6α-ethyl-5β-cholan-24-nor-23-cholanyl)-5-amino-1,3,4-oxadiazole(3)

To a solution of ester 2 (0.50 g, 0.90 mmol) in EtOH (6 mL), hydrazinemonohydrate (65% in water, 0.13 mL, 1.81 mmol) was added and the mixturewas refluxed for 3 hours. The reaction was cooled at room temperatureand cyanogen bromide (0.29 g, 2.71 mmol) was added portionwise. Thesuspension thus obtained was stirred at room temperature for additional5 hours then was quenched by addition of a saturated solution of NaHCO₃(40 mL). The mixture was extracted with AcOEt (3×50 mL), the combinedorganic layers were washed with H₂O (100 mL), brine (100 mL), dried overNa₂SO₄ and evaporated under reduced pressure. The crude was purified byflash chromatography to afford the desired oxadiazolamine derivative 3as colorless oil (0.19 g, 0.34 mmol, 38%).

2-(3α,7α-Dihydroxy-6α-ethyl-5β-cholan-24-nor-23-cholanyl)-5-methylsulfonamido-1,3,4-oxadiazole(Compound 23)

To a solution of oxadiazolamine derivative 3 (0.10 g, 0.18 mmol) inCH₂Cl₂ (10 mL), triethylamine (0.16 mL, 1.10 mmol) and methanesulfonylchloride (0.04 mL, 0.55 mmol) were added and the resulting mixture wasrefluxed for 4 hours. The reaction was then quenched with a saturatedsolution of NH₄Cl (20 mL) and extracted with CH₂Cl₂ (3×10 mL). Thecombined organic layers were washed with a saturated solution of NaHCO₃(30 mL), H₂O (30 mL), brine (30 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue was then dissolved into MeOH (3 mL)and treated with HCl 3 N (1 mL). The solvent was removed under reducedpressure, the residue was dissolved in H₂O (15 mL) and extracted withEt₂O (3×15 mL). The combined organic layers were washed with H₂O (30mL), brine (30 mL), dried over Na₂SO₄ and evaporated under reducedpressure. The crude was purified by flash chromatography eluting to giveCompound 23 as white solid (26 mg, 0.05 mmol, 27%).

Rf=0.31 (SiO₂, F-254, CH₂Cl₂/MeOH 95:5). ¹H-NMR (DMSO-d₆, 400 MHz) δ:0.57 (3H, s, 18-CH₃), 0.79-0.85 (6H, m, 19-CH₃+CH₂CH₃), 0.90 (3H, d,J=5.9 Hz, 21-CH₃), 2.49-2.68 (2H, m, 23-CH₂), 2.93 (3H, s, SCH₃),3.09-3.13 (1H, m, 3-CH), 3.47 (1H, s, 7-CH). ¹³C-NMR (DMSO-d₆, 100.6MHz) δ 12.5 (2×), 18.8, 21.2, 22.5, 22.9, 23.9 (2×), 28.6, 31.1, 31.9,33.5, 34.2, 35.5, 35.9, 36.3, 39.3, 42.1 (2×), 42.9, 46.1, 50.9,56.69.30, 71.5, 157.6, 160.6.

Example 24: Synthesis of Compound 24

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-5-cholan-24-oate (2)

To a solution of OCA (1) (250 mg, 0.59 mmol) in MeOH (10 mL),p-toluensulfonic acid monohydrate (10 mg, 0.06 mmol) was added, and theresulting mixture was sonicated for 2 hours. The solvent was removedunder reduced pressure, the residue was dissolved in AcOEt (15 mL) andsequentially washed with a saturated solution of NaHCO₃ (15 mL), H₂O (10mL) and brine (10 mL). The organic layer was dried over Na₂SO₄ andconcentrated under reduced pressure. The residue was then dissolved inCH₂Cl₂ (15 mL), and to the resulting solution diisopropylethylamine(0.32 mL, 4.23 mmol), 4-(N,N-dimethylamino)-pyridine (7 mg, 0.06 mmol)and methoxymethylchloride (0.27 mL, 3.55 mmol) were sequentially added.The mixture was then refluxed until completeness. The reaction wascooled at room temperature and washed with HCl 3 N (10 mL), H₂O (10 mL),a saturated solution of NaHCO₃ (20 mL) and brine (15 mL). The organiclayer was dried over Na₂SO₄ and concentrated under reduced pressure, toafford 302 mg of 2 as pale yellow oil that was used for the followingstep without further purification (0.3 g, 0.58 mmol).

Methyl3α,7α-dimethoxymethyloxy-6α-ethyl-23(R+S)-hydroxy-5β-cholan-24-oate (3)

To a stirred solution of diisopropylamine (0.65 mL, 4.60 mmol) indistilled THF (5 mL) under N₂ atmosphere and cooled at −40° C., nBuLi(2.5 M in hexane, 1.77 mL, 4.43 mmol) was added dropwise. After 15minutes, the solution was cooled to −78° C. and chlorotrimethylsilane(0.59 mL, 4.72 mmol) was added dropwise. A solution of 2 (0.30 g, 0.58mmol) in THF (5 mL) was added portionwise at −70° C. Once the additionwas finished, the reaction mixture was stirred at −78° C. for 1 hour andthen allowed to react at room temperature. Volatiles were removed underreduced pressure. The residue was directly dissolved in distilled CH₂Cl₂(5 mL). The resulting solution was added dropwise to a suspension offreshly crystallized and acetic acid free lead(IV)tetraacetate (0.38 g,0.56 mmol) in distilled CH₂Cl₂ (7 mL) under N₂ atmosphere. After 30minutes the reaction mixture was filtered under vacuum through a Celitepad. The filtrate was concentrated under reduced pressure and theresidue was filtered through a silica gel pad. The residue was dissolvedin MeOH (6 mL) and treated with potassium carbonate (0.16 g, 1.15 mmol).The resulting suspension was vigorously stirred at room temperature for15 minutes. The mixture was then diluted with CH₂Cl₂ (15 mL) andfiltered under vacuum. The filtrate was further diluted with additionalCH₂Cl₂ (15 mL) and washed with brine (20 mL). The aqueous phase wasextracted with CH₂Cl₂ (3×10 mL), and all the collected organic layerswere dried over Na₂SO₄ and concentrated under reduced pressure. Theresidue was purified by flash chromatography to afford 0.13 g of 3 asmixture of two C23-epimers (0.13 g, 0.24 mmol, 41%).

Methyl3α,7α-dimethoxymethyloxy-6α-ethyl-23(R+S)-(methanesulfonyloxy)-5β-cholan-24-oate(4)

To a stirred solution of 3 (0.13 g, 0.24 mmol) in pyridine (5 mL),methanesulfonyl chloride (0.09 mL, 1.18 mmol) was added, and theresulting mixture was stirred at room temperature for 12 hours. Themixture was then poured into H₂O (10 mL) and extracted with CH₂Cl₂ (3×10mL). The collected organic layers were washed with HCl 0.5 M (3×5 mL),with a saturated solution of NaHCO₃ (10 mL), brine (10 mL), dried overNa₂SO₄ and concentrated under reduced pressure, to give 4 (as mixture oftwo C23-epimers) which was used such as for the next step.

Methyl 3α,7α-dimethoxymethyloxy-6α-ethyl-23(R+S)-bromo-5β-cholan-24-oate(5)

To a solution of 4 (0.24 mmol) in DMF (5 mL), lithium bromide (0.06 g,0.71 mmol) was added, and the resulting mixture was stirred at 40° C.for 6 hours. AcOEt (10 mL) was then added, and the mixture was washedwith H₂O (3×10 mL), brine (10 mL), dried over Na₂SO₄ and concentratedunder reduced pressure. The residue was purified to afford 90 mg ofdesired bromo derivative 5 as mixture of two C23-epimers (0.09 g, 0.15mmol, 63% from 3).

3α,7α-Dimethoxymethyloxy-6α-ethyl-23,24-bisnor-22-(2-imino-4-oxo-thiazolidin-5-yl)-5β-cholane(6)

To a solution of bromo derivative 5 (90 mg, 0.15 mmol) in EtOH (10 mL),thiourea (91 mg, 1.19 mmol) and sodium acetate (98 mg, 1.19 mmol) wereadded, and the resulting mixture was stirred and refluxed for 24 hours.The reaction was cooled at room temperature and volatiles were removedunder reduced pressure. The residue was dissolved in AcOEt (10 mL),washed with H₂O (2×10 mL), brine (10 mL), dried over Na₂SO₄ andconcentrated under reduced pressure to afford 6 as mixture of epimers.The crude was used for the next step without further purification.

3α,7α-Dihydroxy-6α-ethyl-23,24-bisnor-22-(2,4-dioxo-thiazolidin-5-yl)-5β-cholane(Compound 24)

To a solution of iminothiazolidine derivative 6 (0.15 mmol) in EtOH (4mL), HCl 37% (0.7 mL) was added and the resulting mixture was stirredand refluxed. The mixture was then treated with H₂O (8 mL) and organicvolatiles were removed under reduced pressure. H₂O (3 mL) was added andthe mixture was extracted with CH₂Cl₂ (3×7 mL), dried over Na₂SO₄ andconcentrated under reduced pressure. After chromatographic purification,5 mg of desired Compound 24 were obtained (5 mg, 0.01 mmol, 7% from 5).

Rf=0.28 (SiO₂, F-254, CH₂Cl₂/MeOH 90:10). M. p.=136-138° C. ¹H-NMR(CD₃OD, 400 MHz) δ: 0.71 (3H, s, 18-CH₃), 0.87-0.98 (6H, m,19-CH₃+CH₂CH₃), 1.01 (3H, d, J=6.6 Hz, 21-CH₃), 3.29-3.33 (1H, m, 3-CH),3.63 (1H, s, 7-CH), 4.14 (1H, dd, J₁=3.9 Hz, J₂=11.7 Hz, 23-CH), 4.59(1H, bs, NH). ¹³C-NMR (CD₃OD, 100.6 MHz) δ 12.0, 12.2, 18.2, 21.9, 23.5,23.8, 24.5, 29.4, 30.8, 31.2, 34.4, 34.5, 36.6, 36.7, 36.8, 40.9, 41.4,41.5, 43.1, 43.9, 46.9, 51.7, 57.6, 71.1, 73.2, 174.4, 179.6.

Example 25: Physico-Chemical Properties

Critical Micellar Concentration

The detergency was evaluated by calculating the critical micellarconcentration (CMC) with two different methods: surface tension (ST) anddye solubilization (Roda et al. 1983). In the first method, the surfacetension was performed by maximum bubble-pressure method using aSensadyne 6000 tensiometer (Chem-Dyne Research Corp., Milwaukee, Wis.).The surface tension of aqueous solutions at various concentrations(range 0.1-100 mM) of the BA sodium salts in 0.1 M NaCl was measured at25° C. The surface tension values were plotted against the logarithm ofthe bile salt concentration; the regression lines corresponding to thetwo parts of the curve (monomeric and micellar phases) were calculatedusing the method of least squares. The critical micellar concentration(CMC) value (mM) was obtained by the intersection of the two lines.

The second method is based on the fact that some dyes, specificallyOrange OT (purchased from Intercept Pharmaceuticals S.p.a., San Diego,Calif.), are almost insoluble in water but dissolve in solutions withmicellar aggregates that incorporate them; thus, the intensity of colorof the solution increases with bile salt concentration (after CMCachievement). The amount of dye solubilized in relation to bile saltconcentration was determined spectrophotometrically.

For each bile acid, various solution at different concentrations,between 50 mM and 0.1 mM with appropriate dilutions, were incubatedunder stirring at room temperature for 3 days with an excess of OrangeOT. Then all the solutions were centrifuged and filtered through a 0.22μm Millipore fiiter (Millipore Corp., Bedford, Mass.).

Absorbance of each solution was measured at 483 nm (typical wavelengthof Orange OT absorption) with Spectrofotometer (Wellwarm, Labsystems,Cambridge, UK)

Water Solubility

BA were suspended in 100 ml of 0.1 M HCl, pH 1.00, and the saturatedsolutions were transferred to a thermostat-equipped water bathmaintained at 25° C. After incubation for 1 week, the solutions werefiltered on a Millipore filter (0.22 mm), and the concentration of BAwas measured by HPLC-ESMS/MS as reported below.

Lipophilicity

1-Octanol/water partition coefficient was evaluated using a conventionalshake-flask procedure as previously described (Roda et al., 1990). Theexperiments were carried out on 1 mM initial bile salt solution bufferedat pH 8.00 with 0.1 M potassium phosphate buffer to ensure completeionization of the BA. BA concentration in the water phase before andafter partition in 1-octanol was measured by HPLC-ESMS/MS as reportedbelow.

Albumin Binding

Albumin binding was evaluated by equilibrium dialysis at a fixedBA-albumin ratio (Roda et al., 1982). BA was dissolved at aconcentration of 100 mM in 5% bovine serum albumin saline solution andleft to stand for 24 hours at 25° C. Two milliliters of this solutionwas dialyzed in cellulose sacs with a molecular weight cut-off of 12-14kDa (Spectra/Por; Spectrum Medical Industries Inc., Rancho Dominguez,Calif.) against 25 ml of saline solution. The system was equilibrated bymechanical shaking for 72 hours at 25° C. BA concentrations in thestarting solution and in the dialyzed solution were determined byHPLC-ES-MS/MS as reported below.

TABLE 2 Ws CMC ST_(CMC) Albumin BA (μM) (mM) CMpH Dyne/cm LogP_(A) ⁻binding (%) pKa CDCA  32 3.2 7 45.5 2.2 96 5    GCDCA  7 2 6.4 45.2 0.485 3.9  TCDCA hs 3 — 47.1 0.9 70 <1     UDCA  7 10 8.2 50.5 2.2 94 5   CA 273 9 6.5 49 1.1 88 5    TCA hs 4 — 51 −0.5 42 <1     GCA  32 8 6.348.8 −0.4 65 3.9  Ref. Cmpd. C hs 1.3 — 47.9 2.0 85 <1     Ref. Cmpd. B 9 2.9 7.2 48.8 2.5 96 5    Ref. Cmpd. A  99 2 6.1 50.1 1.4 62 5   T-Ref. Cmpd. A hs 1.4 — 47.8 −0.2 81 <1     G-Ref. Cmpd. A 1700  1.3 3.943.8 0.3 71 3.9  Nor-CDCA  23 20 7.9 — 0.5 95 5    Compound 3 225 10 2.7— 1.0 99 1.10* Compound 4 3201  5 4.5 — −0.2 55 4.36* Compound 8 971 63.6 — 0.01 84 2.82* Compound 9 469 6 3.9 — 0.2 89 2.82* Compound 10  168.5 3.7 — 1.4 66 1.10* Compound 11 392 5 7.0 — 1.6 84 5.94* Compound 12517 5 6.6 — 1.5 83 5.59* Compound 14 2025  10 5.0 — 1.9 99 5    Compound15 132 n.d. n.c. — 1.2 76 5.59* Compound 17  5 11 9.0 — 2.0 90 5.71*Compound 19  2151** 5 1.3 — 1.0 51 14.3*  Compound 21 1814  4 14.6 — 0.986 <1*     CDCA: chenodeoxycholic acid GCDCA: glycine conjugate of CDCATCDCA: taurine conjugate of CDCA nor-CDCA: 24-nor-CDCA UDCA:ursodeoxycholic acid CA: cholic acid GCA: glycine conjugate of CA TCA:taurine conjugate of CA Ref. Cmpd. A

T-Ref. Cmpd A: taurine conjugate of Ref. Cmpd A G-Ref. Cmpd A: glycineconjugate of Ref. Cmpd A Ref. Cmpd. C

Ref. Cmpd. B

Example 26: In Vitro TGR5/FXR Activity

Screening Assay

FXR Activity: AlphaScreen Coactivator Recruitment Assay.

Activation of the FXR receptor was determined using a recruitmentcoactivator assay, namely AlphaScreen technology. The assays wereperformed using human glutathione transferase-tagged FXR-LBD (LifeTechnologies, USA) and mouse glutathione transferase-tagged FXR-LBD(generated in-house). Briefly, assays were conducted in white,low-volume, 384-well OptiPlate using a final volume of 25 μL containing10 nM glutathione transferase-tagged FXR-LBD protein and 30 nMbiotinylated Src-1 peptide. The stimulation was carried out withdifferent BA concentrations for 30 minutes at 25° C. Luminescence wasread in an EnVision 2103 microplate analyzer (Perkin Elmer, USA) afterincubation with the detection mix (acceptor and donor beads) for 4 hrsat 25° C. in the dark. Dose-response curves were performed in triplicateand Z′ factor was used to validate the robustness of the assay.

TGR5 Activity: Intracellular cAMP Levels Detection.

Activation of TGR5 was assessed by measuring the level of cAMP using anHTR-FRET assay. Thus, NCI-H716 cells were cultured on 96-well platescoated with Matrigel (Corning, USA) (0.75 mg/ml) in DMEM supplementedwith 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycinsulphate. After 24 hrs, cells were stimulated with increasingconcentrations of test BA for 60′ at 37° C. in OptiMEM (LifeTechnologies, CA, USA) containing 1 mM 3-isobutyl-1-methylxanthine. Thelevel of intracellular cAMP was determined with Lance kit. Z′ factor wasused to validate assays.

hTGR5 CHO-k1 and mTGR5 CHO-Pi10 clone 4 were maintaining in culturemedium: DMEM F12 with 10% FBS, 10 μg/mL puromycine (Sigma Aldrich) andF12 Kaighn's medium with 10% FBS, 600 μg/mL geneticine (Invitrogen), 10μg/mL puromycine (Sigma Aldrich), respectively.

At the day of the experiment, cells were stimulated with differentconcentrations of test compounds dispensed by HP D300 Digital Dispenserfor 30′ at 37° C. according to previous protocol.

Cytotoxicity Assays

Cell viability was evaluated by measuring ATP levels using CellTiter-Glo(Promega), according to the manufacturer's instructions. LCA was used asbile acid comparator for cell cytotoxicity, whereas tamoxifen (Sigma)was used as a control of the assay. Cell necrosis was evaluated bymeasuring the release of lactate dehydrogenase (LDH) from the necroticcells using CytoTox-ONE, a homogeneous membrane integrity assay(Promega), according to manufacturer's instructions. For analyses ofcell viability (ATP levels), apoptosis and necrosis (LDH release), 2×10⁴HepG2 cells were stimulated in MEM (EuroClone) medium with 2 mML-Glutamine (EuroClone), 1% penicillin/streptomycin (EuroClone) and 10%FBS (EuroClone) with test compounds at concentrations ranging from 100nM to 350 μM in a white 96-well microplate for 4 hrs at 37° C.

GLP1 Secretion

Human NCI-H716 cells were seeded into 24-well culture plates precoatedwith Matrigel (BD Bioscences) in DMEM high glucose (EuroClone), 2 mML-Glutamine (EuroClone), 1% penicillin/streptomycin (EuroClone), 10% FBS(EuroClone). Twenty-four hours later, the supernatants were replaced byPBS containing 1 mM CaCl and dipeptidyl peptidase IV inhibitordiprotin-A (Sigma) and stimulated with tested compound for 1 h at 37° C.GLP-1 was measured by Bio-Plex (Bio-Rad Laboratories) and normalized toprotein content.

Biological activities of representative compounds of the application arepresented in Table 3.

TABLE 3 TGR5 hTGR5 CHO hFXR (NCI-H716) (mTGR5 CHO) (mFXR) GLP-1Secretion EC₅₀ Efficacy EC₅₀ Efficacy EC₅₀ Efficacy Fold relative to NTCmpd (μM) (%) (μM) (%) (μM) (%) (%) Ref. LCA 3.2-8 100% at 0.8 ± 0.3100% at — — — 10 μM 10 μM Ref. — — — — 10-20 100% at — CDCA 50 μM Ref. —— — — — — — tamoxifen 1 19  40 3.5 ± 1.5 103 ± 1 32 ± 8   52 — (0.7 ±0.3) (115 ± 2) (146 ± 15)   (9 ± 3) 2 12  55 17 ± 2   47.5 ± 2.5 20 ± 9 200 — (1.5 ± 0.2) (93.5 ± 3)  (84 ± 6)  (61 ± 1) 3 9 65 1.5 ± 0.5   110± 7.3 4.3 ± 1.7 115 ± 40 140 (0.18 ± 0.03)  (110 ± 11) (28.5 ± 0.5)  (69± 1) 4 7.5 ± 5    77 ± 10 4.65 ± 0.5    99 ± 1.4 80 ± 30 20 ± 2 230 ±0.5 (1.3 ± 0.3)  (102.5 ± 0.01) (>150)  5 — —   21 (3.4) 43.6 (84.6) 20 37 — 6 — — 29.5 (3.8) 28 (81) >100  — — 7 — — 7.9 (2)   27 (119) 13  83— 8 7 ± 3   97 ± 1.7 0.5 ± 0.1 103 ± 2 5.6 ± 1   113 ± 3  340 ± 0.8(0.78 ± 0.1)  (100 ± 2) (59.5 ± 2)   (47 ± 7) 9 22 ± 3   75 ± 6 1.4 ±0.3  98 ± 2 2.9 ± 0.9 147 ± 20 270 ± 0.7 (1.8 ± 0.2)  (90 ± 10) (55 ±2)  (127 ± 5)  10 1.6 ± 0.2 125 ± 1 0.34 ± 0.04 103 ± 1  0.2 ± 0.04 128± 4  260 (0.6 ± 0.1) (102 ± 6) (7.1 ± 2)   (143 ± 3)  11 2 105  2.7 ±1.8  96 ± 19  1 140 900 ± 2    (1.4 ± 0.75)   (108 ± 7.8) (7.7 ± 1)  (100 ± 16) 12 4 104  0.71 ± 0.08   101 ± 1.4 1.7 ± 0.6 125 ± 15 420 ±1   (0.84 ± 0.14)   (105 ± 0.7) (12.7 ± 4)   (93 ± 1) 13 6.4 ± 1.1  84 ±14   1 ± 0.1 102 ± 3 0.43 138 — (2.3 ± 0.5)  (90 ± 4) (1.4 ± 0.1) (208 ±2)  14 1.6 ± 0.3 110 ± 2 0.48 ± 0.3  107 ± 1 0.075 ± 0.01  165 ± 15 290± 0.8 (0.9 ± 0.2) (111 ± 2) (0.46 ± 0.04) (264 ± 31) 15 5 ± 1   97 ± 7.60.72 ± 0.05  99 ± 3 0.15 ± 0.05 173 ± 22 — (0.44 ± 0.1)  (102 ± 5) (6 ±2) (222 ± 23) 16   3 ± 0.3 116 ± 9 1.2 ± 0.1  99 ± 4 0.15 ± 0.05 165 ±19 (0.44 ± 0.1)   (99 ± 9) (6.6 ± 1)   (204 ± 25) 17 1.2 ± 0.1 119 ± 2 0.1 ± 0.04 103 ± 2 0.45 ± 0.05 138 ± 9  — (0.8 ± 0.2) (101 ± 5) (2.3 ±0.3) (171 ± 1)  18 1.8 ± 0.2 115 ± 4 0.11 ± 0.03 104 ± 1 0.55 ± 0.03 132± 8  — (0.37 ± 0.1)  (101 ± 6) (2.3 ± 0.3) (150 ± 4)  23 12.5 ± 1.5   75± 1 1.3 ± 0.1 111 ± 2  0.1 ± 0.02 175 ± 22 (1.4 ± 0.4)  (110 ± 10) (0.33± 0.01) (342 ± 35) 19 0.16 ± 0.01 128 ± 1  0.02 ± 0.008 105 ± 2 3 ± 1137 ± 2  500 ± 1   (0.08 ± 0.01) (106 ± 4) (10 ± 1)  (122 ± 2)  20 0.7 ±0.1 148 ± 1  0.3 ± 0.14 109 ± 5  8.7 ± 0.04 122 ± 3  190 ± 0.3  (0.5 ±0.15) (101 ± 4) (16 ± 4)  (85 ± 4) 21 0.19 ± 0.05 127 ± 1 0.02 ± 0.01105 ± 2 1.5 ± 0.5 136 ± 7  450 ± 0.5 (0.07 ± 0.03) (107 ± 3)  (5.4 ±0.02) (136 ± 5) 

Example 27: Pharmacokinetic Properties after Oral Administration at 30mg/kg to ob/ob Mouse

Pharmacokinetics studies were performed in male ob/ob mice (9-10 wk-old,Janvier/Charles River Laboratories). Mice were orally dosed withcompounds (30 mg/kg suspension in 0.5% hydroxyethylcellulose). Blood wassampled 10, 30 min and 1, 2, 4, 6 and 24 hours after administration intoLithium-Heparin tubes. Plasma was collected upon centrifugation andfrozen for further measurements. The plasma concentration of thecompound was determined using a HPLC-ESI-MS/MS method following anon-line extraction (Turboflow). The MS system (Sciex API4000) was setwith an electrospray ionization source (Turbospray) in the negative modewith optimized parameters. Chromatograms were acquired using the massspectrometer in multiple reaction monitoring mode.

Example 28: OGTT in 3-Day Treated ob/ob Mice

Male ob/ob mice (10 wk-old, Janvier, n=9 per group) were orally treatedtwice a day (BID) for 3 days with vehicle (0.5% HEC) or the BA understudy (100 mg/kg). Immediately after the last administration, mice werefasted for 4 hours. Then an oral glucose tolerance test was performed(glucose 1.5 g/kg). Blood samples were collected at TO (before glucoseadministration), 10, 25, 60 and 120 minutes for blood glucose levelsdetermination using a glucometer and at T0 (before glucoseadministration), 10, 25 and 60 minutes for plasma insulin levelmeasurement (ALPCO ELISA kit). At the end of the experiments, bilevolume within the gallbladder was determined in anesthetized mice.

Example 29: In Vivo GLP-1 Secretion in Normal Mice

Male C57Bl/6 mice (9 wk-old, Janvier) were overnight fasted and thenorally treated with the BA under study (100 mg/kg), followed bysitagliptin (1 mg/kg). The time between administration of the BA understudy and sitagliptin varies between 0 and 3 hours, depending on the PKprofile of the BA under study. One hour after sitagliptin treatment,mice were orally challenged with glucose (1.5 g/kg). Before and 5minutes after glucose challenge, blood was collected for blood glucosedetermination using a glucometer (AccuChek) and for plasma recoveryusing K₃-EDTA tubes containing DPP-IV inhibitor. Plasma levels of activeGLP1 were measured by ELISA according to manufacturer's instructions(Linco-Millipore). At the end of the experiments, bile volume within thegallbladder was determined in anesthetized mice.

Example 30: Pharmacokinetic Study in “Bile Fistula Rat” Model

Bile fistula rat model was reported in Roda et al., 2014, J. PharmacolExp Ther, Supplemental Data IV. Briefly, after animals wereanesthetized, the bile duct was cannulated, and the BAs were deliveredeither intravenously or intraduodenally per gavage. Each bile acid wasinfused at a dose of 1 mmol/min/kg body weight over 1 hour at 2.5ml/hour. Bile was collected at 15-minute time intervals throughout theinfusion and over 2 hours after the infusion was over. Plasma wascollected at 30-minute time intervals throughout the intraduodenalinfusion and over 2 hours after the infusion was over while for theintravenous infusion plasma samples were collected at beginning and atthe end of experiment. Liver and intestinal content were collected atthe end of each experiment.

HPLC-ES-MS/MS Method

As previously reported (Roda et al., 2014, J Pharmacol Exp Ther), BAswere separated in elution gradient mode using 15 mM ammonium acetatebuffer (pH 8.00) as mobile phase A and acetonitrile/methanol (75:25 v/v)as mobile phase B. The MS system was set with an electrospray ionizationsource (ES) in the negative mode with optimized parameters.Chromatograms were acquired using the mass spectrometer in multiplereaction monitoring mode.

Bile Sample Preparation

Rat bile samples were brought to 25° C. and diluted 1:100 or 1:10 (v/v)with ammonium acetate buffer 15 mM, pH 8.00, and acetonitrile/methanol(3:1 v/v) in ratio 65:35 (v/v). The final solution was transferred to anautosampler vial, and 5 ml was injected onto the column. The bilesectretion flow results are expressed as μmol/kg/min while the bile flowresults are expressed as μL/kg/min.

Plasma Sample Preparation.

As previously reported (Roda et al., 2014, J Pharmacol Exp Ther), plasmasamples (100 ml) were diluted 1:6 (v/v) with 0.1 N NaOH and heated to64° C. for 30 minutes. The solid phase extraction (SPE) C18 cartridgewas conditioned with 5 ml of methanol and 5 ml of water prior to sampleloading. Plasma samples were loaded into the conditioned cartridge andthen washed with 10 ml of water. The cartridge was then eluted with 5 mlof methanol, the eluate was dried under vacuum and then reconstitutedwith 200 ml of the mobile phase, and 5 μl was injected into theHPLC-ES-MS/MS instrument. The results are expressed as μM.

Liver Sample Preparation.

As previously reported (Roda et al., 2014, J Pharmacol Exp Ther),aliquots weighing approximately 1 g each were taken from differentpoints of the liver sample. Each aliquot was weighed, and 2 ml ofphosphate buffer (0.005 M, pH 7.2) was added. The mixture washomogenized using a potter, which was then washed with methanol (3×1ml). The mixture was sonicated for 5 minutes, vortexed for 2 minutes,heated to 37° C. for 20 minutes, and centrifuged (2100 g for 15minutes). One milliliter of the supernatant was spiked with 10 ml of theinternal standard working solution and dried under vacuum. The residuethen was resuspended with 2 ml of NaOH (0.1 N). The solution wassonicated for 10 minutes, heated to 64° C. for 30 minutes, and SPE wascarried out on C18 extraction cartridges (as shown above). The eluatewas dried under vacuum and reconstituted with 200 ml of the mobile phaseand injected into the HPLC-ES-MS system. The results are expressed asμmol/g where g is total liver weight.

Intestinal Content Sample Preparation

As previously reported (Roda et al., 2014, J Pharmacol Exp Ther),intestinal content sample sample was collected and homogenized using amixer. Aliquots weighing approximately 1 g were taken from thehomogenate. Each aliquot was weighed, and 3 ml of isopropyl alcohol wasadded. The mixture was vortexed for 2 minutes and centrifuged (2100 gfor 10 minutes). The supernatant was then diluted 1:100 v/v with mobilephase, and 190 ml of these final solutions were spiked with 10 ml ofinternal standard. The results are expressed as mol/g where g is totalintestinal content weight.

Calibration Curve

Calibration curve of bile, stool and liver sampled was performed inmobile phase, linearity range 0.1-20 nM. For plasma sample, thecalibration curve was obtained using BA free rat plasma in linearityrange 0.1-20 nM.

Recovery %

The recovery % was evaluated comparing the amount of BA under study andits metabolites in each matrix with the total amount of BA administered.

Other Embodiments

While the application has been described in conjunction with thedetailed description thereof, the foregoing description is intended toillustrate and not limit the scope of the application, which is definedby the scope of the appended claims. Other aspects, advantages, andmodifications are within the scope of the following claims. It will beunderstood by those skilled in the art that various changes in form anddetails may be made therein without departing from the scope of theapplication encompassed by the appended claims.

The invention claimed is:
 1. A compound of formula A:

or a pharmaceutically acceptable salt, ester, tautomer, or amino acidconjugate, thereof, wherein: A is

oxadiazolonyl, or isoxazolonyl, wherein the carbon atom marked with “*”is bonded to the carbon atom to which A is bonded; n is 0, 1, or 2; R₁is H or OH; R₂ is H or OH; R₃ is CR₁₁R₁₂C(O)OH, C(O)NHR₃₁, tetrazolyl,oxadiazolyl, oxadiazolonyl, or thiazolidine-dionyl optionallysubstituted with NHS(O)₂—(C₁-C₃)alkyl; R₁₁ and R₁₂ are eachindependently H, F, OH, CH₂OH, or CH₂F, provided that R₁₁ and R₁₂ arenot both H; R₃₁ is OH, (CH₂)_(p)OH, or (CH₂)_(p)OSO₃H; and p is 1 or 2.2. The compound of claim 1, wherein A is oxadiazolonyl or isoxazolonyl.3. The compound of claim 1, wherein A is


4. The compound of claim 1, wherein R₁ and R₂ are each H.
 5. Thecompound of claim 1, wherein R₁ is H, and R₂ is OH.
 6. The compound ofclaim 1, wherein R₂ is H, and R₁ is OH.
 7. The compound of claim 1,wherein R₁ and R₂ are each OH.
 8. The compound of claim 1, wherein R₃ isCR₁₁R₁₂C(O)OH.
 9. The compound of claim 1, wherein n is
 1. 10. Thecompound of claim 1, wherein R₃ is C(O)NHR₃₁.
 11. The compound of claim10, wherein R₃₁ is OH.
 12. The compound of claim 10, wherein R₃₁ is(CH₂)_(p)OH.
 13. The compound of claim 10, wherein R₃₁ is(CH₂)_(p)OSO₃H.
 14. The compound of claim 1, wherein the compound is offormula I:

or a pharmaceutically acceptable salt, ester, tautomer, or amino acidconjugate thereof, wherein: R₁₁ and R₁₂ are each independently H, F, OH,CH₂OH, or CH₂F, provided that R₁₁ and R₁₂ are not both H; and R₁₃ is Hor OH.
 15. The compound of claim 1, wherein the compound is of formulaII:

or a pharmaceutically acceptable salt, ester, tautomer, or amino acidconjugate thereof, wherein: q is 0, 1, or 2; R₂₁ and R₂₂ are eachindependently H or OH; and R₂₃ is tetrazolyl, oxadiazolyl,oxadiazolonyl, or thiazolidine-dionyl optionally substituted withNHS(O)₂—(C₁-C₃)alkyl.
 16. The compound of claim 1, wherein the compoundis of formula III:

or a pharmaceutically acceptable salt, ester, tautomer, or amino acidconjugate thereof, wherein: R₃₁ is OH, (CH₂)_(p)OH, or (CH₂)_(p)OSO₃H;and p is 1 or
 2. 17. A pharmaceutical composition comprising thecompound of claim 1, and at least one pharmaceutically acceptableexcipient.